Inhibitors of bruton&#39;s tyrosine kinase for the treatment of solid tumors

ABSTRACT

Described herein are irreversible Btk inhibitor compounds, and methods for using such irreversible inhibitors in the treatment of diseases and disorders characterized by the presence or development of solid tumors.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/793,366, filed Jul. 7, 2015; which is a continuation of U.S.application Ser. No. 13/965,135, filed Aug. 12, 2013, now U.S. Pat. No.9,107,924, issued Oct. 24, 2017; which is a continuation-in-part of U.S.application Ser. No. 13/341,708, filed on Dec. 30, 2011, now U.S. Pat.No. 8,883,803, issued Nov. 11, 2014; and U.S. application Ser. No.13/341,695, filed on Dec. 30, 2011, now U.S. Pat. No. 9,278,100, issuedMar. 8, 2016, which are both continuations of U.S. application Ser. No.13/003,811, filed on May 25, 2011; which is a U.S. National Stage Entryof PCT/US2009/050897, filed Jul. 16, 2009; which claims the benefit ofU.S. Provisional Application No. 61/081,344, filed on Jul. 16, 2008; allof which are herein incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 23, 2017, isnamed PIR-78207 Sequence.txt and is 2,917 bytes in size.

BACKGROUND OF THE INVENTION

A kinase, alternatively known as a phosphotransferase, is a type ofenzyme that transfers phosphate groups from high-energy donor molecules,such as ATP, to specific target molecules; the process is termedphosphorylation. Protein kinases, which act on and modify the activityof specific proteins, are used to transmit signals and control complexprocesses in cells. Up to 518 different kinases have been identified inhumans. Their enormous diversity and role in signaling makes themattractive targets for drug design.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are methods for treating HER2amplified breast cancer in an individual in need thereof comprisingadministering to an individual in need thereof a composition comprisinga therapeutically-effective amount of a compound of Formula (C1) havingthe structure:

wherein:

-   -   Y is an optionally substituted group selected from among alkyl,        heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, and        alkylheterocycloalkyl;    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring;    -   G is

where R^(a) is H, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl; and either

-   -   R₇ and R₈ are H;    -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₆ and R₈ are H;    -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₇ and R₈ taken together form a bond;    -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).        In some embodiments,    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring; and    -   Z is C(═O).        In some embodiments,    -   R₆ and R₈ are H; and    -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).        In some embodiments,    -   Y and R₁₂ taken together form a 6-membered heterocyclic ring;        and    -   R₆ and R₈ are H; and    -   R₇ is substituted or unsubstituted H, substituted or        unsubstituted C₁-C₄alkyl, substituted or unsubstituted        C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).        In some embodiments, the compound of Formula (I) is        (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

In some embodiments, the method further comprises administering anadditional therapeutic agent selected from: trastuzumab, paclitaxel,doxorubicin, cyclophosphamide, and cisplatin. In some embodiments, themethod further comprises administering trastuzumab.

Disclosed herein, in certain embodiment, are methods for treating HER2amplified cancer in an individual in need thereof comprisingadministering to an individual in need thereof a composition comprisinga therapeutically-effective amount of a compound of Formula (C1) havingthe structure:

wherein:

-   -   Y is an optionally substituted group selected from among alkyl,        heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, and        alkylheterocycloalkyl;    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring;    -   G is

where R^(a) is H, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl; and either

-   -   R₇ and R₈ are H;    -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₆ and R₈ are H;    -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₇ and R₈ taken together form a bond;    -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).        In some embodiments,    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring; and    -   Z is C(═O).        In some embodiments,    -   R₆ and R₈ are H; and    -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).        In some embodiments,    -   Y and R₁₂ taken together form a 6-membered heterocyclic ring;    -   R₆ and R₈ are H; and    -   R₇ is substituted or unsubstituted H, substituted or        unsubstituted C₁-C₄alkyl, substituted or unsubstituted        C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).        In some embodiments, the compound of Formula (I) is        (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

In some embodiments, the method further comprises administering anadditional therapeutic agent selected from: trastuzumab, paclitaxel,doxorubicin, cyclophosphamide, and cisplatin. In some embodiments, themethod further comprises administering trastuzumab.

Other objects, features and advantages of the methods and compositionsdescribed herein will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only. The section headingsused herein are for organizational purposes only and are not to beconstrued as limiting the subject matter described.

Certain Terminology

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of any subject matter claimed. In this application,the use of the singular includes the plural unless specifically statedotherwise. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

Definition of standard chemistry terms are found in reference works,including Carey and Sundberg “ADVANCED ORGANIC CHEMISTRY 4^(TH) ED.”Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwiseindicated, conventional methods of mass spectroscopy, NMR, HPLC, proteinchemistry, biochemistry, recombinant DNA techniques and pharmacology,within the skill of the art are employed. Unless specific definitionsare provided, the nomenclature employed in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those known in the art. Standard techniques are optionallyused for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.Standard techniques are optionally used for recombinant DNA,oligonucleotide synthesis, and tissue culture and transformation (e.g.,electroporation, lipofection). Reactions and purification techniques areperformed using documented methodologies or as described herein.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, celllines, constructs, and reagents described herein and as such optionallyvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the methods and compositions describedherein, which will be limited only by the appended claims.

Unless stated otherwise, the terms used for complex moieties (i.e.,multiple chains of moieties) are to be read equivalently either fromleft to right or right to left. For example, the groupalkylenecycloalkylene refers both to an alkylene group followed by acycloalkylene group or as a cycloalkylene group followed by an alkylenegroup.

The suffix “ene” appended to a group indicates that such a group is adiradical. By way of example only, a methylene is a diradical of amethyl group, that is, it is a —CH₂— group; and an ethylene is adiradical of an ethyl group, i.e., —CH₂CH₂—.

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkylmoiety includes a “saturated alkyl” group, which means that it does notcontain any alkene or alkyne moieties. The alkyl moiety also includes an“unsaturated alkyl” moiety, which means that it contains at least onealkene or alkyne moiety. An “alkene” moiety refers to a group that hasat least one carbon-carbon double bond, and an “alkyne” moiety refers toa group that has at least one carbon-carbon triple bond. The alkylmoiety, whether saturated or unsaturated, includes branched, straightchain, or cyclic moieties. Depending on the structure, an alkyl groupincludes a monoradical or a diradical (i.e., an alkylene group), and ifa “lower alkyl” having 1 to 6 carbon atoms.

As used herein, C₁-C_(x) includes C₁-C₂, C₁-C₃ . . . C₁-C_(x).

The “alkyl” moiety optionally has 1 to 10 carbon atoms (whenever itappears herein, a numerical range such as “1 to 10” refers to eachinteger in the given range; e.g., “1 to 10 carbon atoms” means that thealkyl group is selected from a moiety having 1 carbon atom, 2 carbonatoms, 3 carbon atoms, etc., up to and including 10 carbon atoms,although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated). The alkyl group of thecompounds described herein may be designated as “C₁-C₄ alkyl” or similardesignations. By way of example only, “C₁-C₄ alkyl” indicates that thereare one to four carbon atoms in the alkyl chain, i.e., the alkyl chainis selected from among methyl, ethyl, propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, and t-butyl. Thus C₁-C₄ alkyl includes C₁-C₂ alkyland C₁-C₃ alkyl. Alkyl groups are optionally substituted orunsubstituted. Typical alkyl groups include, but are in no way limitedto, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like.

The term “alkenyl” refers to a type of alkyl group in which the firsttwo atoms of the alkyl group form a double bond that is not part of anaromatic group. That is, an alkenyl group begins with the atoms—C(R)═C(R)—R, wherein R refers to the remaining portions of the alkenylgroup, which are either the same or different. The alkenyl moiety isoptionally branched, straight chain, or cyclic (in which case, it isalso known as a “cycloalkenyl” group). Depending on the structure, analkenyl group includes a monoradical or a diradical (i.e., an alkenylenegroup). Alkenyl groups are optionally substituted. Non-limiting examplesof an alkenyl group include —CH═CH₂, —C(CH₃)═CH₂, —CH═CHCH₃,—C(CH₃)═CHCH₃. Alkenylene groups include, but are not limited to,—CH═CH—, —C(CH₃)=CH—, —CH═CHCH₂—, —CH═CHCH₂CH₂— and —C(CH₃)═CHCH₂—.Alkenyl groups optionally have 2 to 10 carbons, and if a “lower alkenyl”having 2 to 6 carbon atoms.

The term “alkynyl” refers to a type of alkyl group in which the firsttwo atoms of the alkyl group form a triple bond. That is, an alkynylgroup begins with the atoms —C≡C—R, wherein R refers to the remainingportions of the alkynyl group, which is either the same or different.The “R” portion of the alkynyl moiety may be branched, straight chain,or cyclic. Depending on the structure, an alkynyl group includes amonoradical or a diradical (i.e., an alkynylene group). Alkynyl groupsare optionally substituted. Non-limiting examples of an alkynyl groupinclude, but are not limited to, —C≡CH, —C≡CCH₃, —C≡CCH₂CH₃, —C≡C—, and—C≡CCH₂—. Alkynyl groups optionally have 2 to 10 carbons, and if a“lower alkynyl” having 2 to 6 carbon atoms.

An “alkoxy” group refers to a (alkyl)O— group, where alkyl is as definedherein.

“Hydroxyalkyl” refers to an alkyl radical, as defined herein,substituted with at least one hydroxy group. Non-limiting examples of ahydroxyalkyl include, but are not limited to, hydroxymethyl,2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl,1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl,4-hydroxybutyl, 2,3-dihydroxypropyl, 1-(hydroxymethyl)-2-hydroxyethyl,2,3-dihydroxybutyl, 3,4-dihydroxybutyl and2-(hydroxymethyl)-3-hydroxypropyl.

“Alkoxyalkyl” refers to an alkyl radical, as defined herein, substitutedwith an alkoxy group, as defined herein.

The term “alkylamine” refers to the —N(alkyl)_(x)H_(y) group, where xand y are selected from among x=1, y=1 and x=2, y=0. When x=2, the alkylgroups, taken together with the N atom to which they are attached,optionally form a cyclic ring system.

“Alkylaminoalkyl” refers to an alkyl radical, as defined herein,substituted with an alkylamine, as defined herein.

“Hydroxyalkylaminoalkyl” refers to an alkyl radical, as defined herein,substituted with an alkylamine, and alkylhydroxy, as defined herein.

“Alkoxyalkylaminoalkyl” refers to an alkyl radical, as defined herein,substituted with an alkylamine and substituted with an alkylalkoxy, asdefined herein.

An “amide” is a chemical moiety with the formula —C(O)NHR or —NHC(O)R,where R is selected from among alkyl, cycloalkyl, aryl, heteroaryl(bonded through a ring carbon) and heteroalicyclic (bonded through aring carbon). In some embodiments, an amide moiety forms a linkagebetween an amino acid or a peptide molecule and a compound describedherein, thereby forming a prodrug. Any amine, or carboxyl side chain onthe compounds described herein can be amidified. The procedures andspecific groups to make such amides are found in sources such as Greeneand Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., John Wiley& Sons, New York, N.Y., 1999, which is incorporated herein by referencefor this disclosure.

The term “ester” refers to a chemical moiety with formula —COOR, where Ris selected from among alkyl, cycloalkyl, aryl, heteroaryl (bondedthrough a ring carbon) and heteroalicyclic (bonded through a ringcarbon). Any hydroxy, or carboxyl side chain on the compounds describedherein can be esterified. The procedures and specific groups to makesuch esters are found in sources such as Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3^(rd) Ed., John Wiley & Sons, New York,N.Y., 1999, which is incorporated herein by reference for thisdisclosure.

As used herein, the term “ring” refers to any covalently closedstructure. Rings include, for example, carbocycles (e.g., aryls andcycloalkyls), heterocycles (e.g., heteroaryls and non-aromaticheterocycles), aromatics (e.g. aryls and heteroaryls), and non-aromatics(e.g., cycloalkyls and non-aromatic heterocycles). Rings can beoptionally substituted. Rings can be monocyclic or polycyclic.

As used herein, the term “ring system” refers to one, or more than onering.

The term “membered ring” can embrace any cyclic structure. The term“membered” is meant to denote the number of skeletal atoms thatconstitute the ring. Thus, for example, cyclohexyl, pyridine, pyran andthiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, andthiophene are 5-membered rings.

The term “fused” refers to structures in which two or more rings shareone or more bonds.

The term “carbocyclic” or “carbocycle” refers to a ring wherein each ofthe atoms forming the ring is a carbon atom. Carbocycle includes aryland cycloalkyl. The term thus distinguishes carbocycle from heterocycle(“heterocyclic”) in which the ring backbone contains at least one atomwhich is different from carbon (i.e. a heteroatom). Heterocycle includesheteroaryl and heterocycloalkyl. Carbocycles and heterocycles can beoptionally substituted.

The term “aromatic” refers to a planar ring having a delocalizedπ-electron system containing 4n+2 π electrons, where n is an integer.Aromatic rings can be formed from five, six, seven, eight, nine, or morethan nine atoms. Aromatics can be optionally substituted. The term“aromatic” includes both carbocyclic aryl (e.g., phenyl) andheterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g.,pyridine). The term includes monocyclic or fused-ring polycyclic (i.e.,rings which share adjacent pairs of carbon atoms) groups.

As used herein, the term “aryl” refers to an aromatic ring wherein eachof the atoms forming the ring is a carbon atom. Aryl rings can be formedby five, six, seven, eight, nine, or more than nine carbon atoms. Arylgroups can be optionally substituted. Examples of aryl groups include,but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl,fluorenyl, and indenyl. Depending on the structure, an aryl group can bea monoradical or a diradical (i.e., an arylene group).

An “aryloxy” group refers to an (aryl)O— group, where aryl is as definedherein.

The term “carbonyl” as used herein refers to a group containing a moietyselected from the group consisting of —C(O)—, —S(O)—, —S(O)2-, and—C(S)—, including, but not limited to, groups containing a least oneketone group, and/or at least one aldehyde group, and/or at least oneester group, and/or at least one carboxylic acid group, and/or at leastone thioester group. Such carbonyl groups include ketones, aldehydes,carboxylic acids, esters, and thioesters. In some embodiments, suchgroups are a part of linear, branched, or cyclic molecules.

The term “cycloalkyl” refers to a monocyclic or polycyclic radical thatcontains only carbon and hydrogen, and is optionally saturated,partially unsaturated, or fully unsaturated. Cycloalkyl groups includegroups having from 3 to 10 ring atoms. Illustrative examples ofcycloalkyl groups include the following moieties:

and the like. Depending on the structure, a cycloalkyl group is either amonoradical or a diradical (e.g., an cycloalkylene group), and if a“lower cycloalkyl” having 3 to 8 carbon atoms.

“Cycloalkylalkyl” means an alkyl radical, as defined herein, substitutedwith a cycloalkyl group. Non-limiting cycloalkylalkyl groups includecyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, and the like.

The term “heterocycle” refers to heteroaromatic and heteroalicyclicgroups containing one to four heteroatoms each selected from O, S and N,wherein each heterocyclic group has from 4 to 10 atoms in its ringsystem, and with the proviso that the ring of said group does notcontain two adjacent O or S atoms. Herein, whenever the number of carbonatoms in a heterocycle is indicated (e.g., C₁-C₆ heterocycle), at leastone other atom (the heteroatom) must be present in the ring.Designations such as “C₁-C₆ heterocycle” refer only to the number ofcarbon atoms in the ring and do not refer to the total number of atomsin the ring. It is understood that the heterocylic ring can haveadditional heteroatoms in the ring. Designations such as “4-6 memberedheterocycle” refer to the total number of atoms that are contained inthe ring (i.e., a four, five, or six membered ring, in which at leastone atom is a carbon atom, at least one atom is a heteroatom and theremaining two to four atoms are either carbon atoms or heteroatoms). Inheterocycles that have two or more heteroatoms, those two or moreheteroatoms can be the same or different from one another. Heterocyclescan be optionally substituted. Binding to a heterocycle can be at aheteroatom or via a carbon atom. Non-aromatic heterocyclic groupsinclude groups having only 4 atoms in their ring system, but aromaticheterocyclic groups must have at least 5 atoms in their ring system. Theheterocyclic groups include benzo-fused ring systems. An example of a4-membered heterocyclic group is azetidinyl (derived from azetidine). Anexample of a 5-membered heterocyclic group is thiazolyl. An example of a6-membered heterocyclic group is pyridyl, and an example of a10-membered heterocyclic group is quinolinyl. Examples of non-aromaticheterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl andquinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl,imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl,furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, andfuropyridinyl. The foregoing groups, as derived from the groups listedabove, are optionally C-attached or N-attached where such is possible.For instance, a group derived from pyrrole includes pyrrol-1-yl(N-attached) or pyrrol-3-yl (C-attached). Further, a group derived fromimidazole includes imidazol-1-yl or imidazol-3-yl (both N-attached) orimidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached). Theheterocyclic groups include benzo-fused ring systems and ring systemssubstituted with one or two oxo (═O) moieties such as pyrrolidin-2-one.Depending on the structure, a heterocycle group can be a monoradical ora diradical (i.e., a heterocyclene group).

The terms “heteroaryl” or, alternatively, “heteroaromatic” refers to anaromatic group that includes one or more ring heteroatoms selected fromnitrogen, oxygen and sulfur. An N-containing “heteroaromatic” or“heteroaryl” moiety refers to an aromatic group in which at least one ofthe skeletal atoms of the ring is a nitrogen atom. Illustrative examplesof heteroaryl groups include the following moieties:

and the like. Depending on the structure, a heteroaryl group can be amonoradical or a diradical (i.e., a heteroarylene group).

As used herein, the term “non-aromatic heterocycle”, “heterocycloalkyl”or “heteroalicyclic” refers to a non-aromatic ring wherein one or moreatoms forming the ring is a heteroatom. A “non-aromatic heterocycle” or“heterocycloalkyl” group refers to a cycloalkyl group that includes atleast one heteroatom selected from nitrogen, oxygen and sulfur. In someembodiments, the radicals are fused with an aryl or heteroaryl.Heterocycloalkyl rings can be formed by three, four, five, six, seven,eight, nine, or more than nine atoms. Heterocycloalkyl rings can beoptionally substituted. In certain embodiments, non-aromaticheterocycles contain one or more carbonyl or thiocarbonyl groups suchas, for example, oxo- and thio-containing groups. Examples ofheterocycloalkyls include, but are not limited to, lactams, lactones,cyclic imides, cyclic thioimides, cyclic carbamates,tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin,1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane,1,4-oxathiin, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine,maleimide, succinimide, barbituric acid, thiobarbituric acid,dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane,hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran,pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline,pyrazolidine, imidazoline, imidazolidine, 1,3-dioxole, 1,3-dioxolane,1,3-dithiole, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline,oxazolidine, oxazolidinone, thiazoline, thiazolidine, and1,3-oxathiolane. Illustrative examples of heterocycloalkyl groups, alsoreferred to as non-aromatic heterocycles, include:

and the like. The term heteroalicyclic also includes all ring forms ofthe carbohydrates, including but not limited to the monosaccharides, thedisaccharides and the oligosaccharides. Depending on the structure, aheterocycloalkyl group can be a monoradical or a diradical (i.e., aheterocycloalkylene group).

The term “halo” or, alternatively, “halogen” or “halide” means fluoro,chloro, bromo and iodo.

The term “haloalkyl,” refers to alkyl structures in which at least onehydrogen is replaced with a halogen atom. In certain embodiments inwhich two or more hydrogen atoms are replaced with halogen atoms, thehalogen atoms are all the same as one another. In other embodiments inwhich two or more hydrogen atoms are replaced with halogen atoms, thehalogen atoms are not all the same as one another.

The term “fluoroalkyl,” as used herein, refers to alkyl group in whichat least one hydrogen is replaced with a fluorine atom. Examples offluoroalkyl groups include, but are not limited to, —CF₃, —CH₂CF₃,—CF₂CF₃, —CH₂CH₂CF₃ and the like.

As used herein, the term “heteroalkyl” refers to optionally substitutedalkyl radicals in which one or more skeletal chain atoms is aheteroatom, e.g., oxygen, nitrogen, sulfur, silicon, phosphorus orcombinations thereof. The heteroatom(s) are placed at any interiorposition of the heteroalkyl group or at the position at which theheteroalkyl group is attached to the remainder of the molecule. Examplesinclude, but are not limited to, —CH₂—O—CH₃, —CH₂—CH₂—O—CH₃,—CH₂—NH—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—N(CH₃)—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. In addition, in some embodiments, up to twoheteroatoms are consecutive, such as, by way of example, —CH₂—NH—OCH₃and —CH₂—O—Si(CH₃)₃.

The term “heteroatom” refers to an atom other than carbon or hydrogen.Heteroatoms are typically independently selected from among oxygen,sulfur, nitrogen, silicon and phosphorus, but are not limited to theseatoms. In embodiments in which two or more heteroatoms are present, thetwo or more heteroatoms can all be the same as one another, or some orall of the two or more heteroatoms can each be different from theothers.

The term “bond” or “single bond” refers to a chemical bond between twoatoms, or two moieties when the atoms joined by the bond are consideredto be part of larger sub structure.

The term “moiety” refers to a specific segment or functional group of amolecule. Chemical moieties are often recognized chemical entitiesembedded in or appended to a molecule.

A “thioalkoxy” or “alkylthio” group refers to a —S-alkyl group.

A “SH” group is also referred to either as a thiol group or a sulfhydrylgroup.

The term “optionally substituted” or “substituted” means that thereferenced group may be substituted with one or more additional group(s)individually and independently selected from alkyl, cycloalkyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, alkylthio,arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone,cyano, halo, acyl, nitro, haloalkyl, fluoroalkyl, amino, including mono-and di-substituted amino groups, and the protected derivatives thereof.By way of example an optional substituents may be L_(s)R_(s), whereineach L_(s) is independently selected from a bond, —O—, —C(═O)—, —S—,—S(═O)—, —S(═O)₂—, —NH—, —NHC(O)—, —C(O)NH—, S(═O)₂NH—, —NHS(═O)₂,—OC(O)NH—, —NHC(O)O—, -(substituted or unsubstituted C₁-C₆ alkyl), or-(substituted or unsubstituted C₂-C₆ alkenyl); and each R_(s) isindependently selected from H, (substituted or unsubstitutedC₁-C₄alkyl), (substituted or unsubstituted C₃-C₆cycloalkyl), heteroaryl,or heteroalkyl. The protecting groups that forms the protectivederivatives of the above substituents include those found in sourcessuch as Greene and Wuts, above.

The term “Michael acceptor moiety” refers to a functional group that canparticipate in a Michael reaction, wherein a new covalent bond is formedbetween a portion of the Michael acceptor moiety and the donor moiety.The Michael acceptor moiety is an electrophile and the “donor moiety” isa nucleophile. The “G” groups presented in any of Formula (A1-A6),Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I), orFormula (VII) are non-limiting examples of Michael acceptor moieties.

The term “nucleophile” or “nucleophilic” refers to an electron richcompound, or moiety thereof. An example of a nucleophile includes, butin no way is limited to, a cysteine residue of a molecule, such as, forexample Cys 481 of Btk.

The term “electrophile”, or “electrophilic” refers to an electron pooror electron deficient molecule, or moiety thereof. Examples ofelectrophiles include, but in no way are limited to, Michael acceptormoieties.

The term “acceptable” or “pharmaceutically acceptable”, with respect toa formulation, composition or ingredient, as used herein, means havingno persistent detrimental effect on the general health of the subjectbeing treated or does not abrogate the biological activity or propertiesof the compound, and is relatively nontoxic.

As used herein, the term “agonist” refers to a compound, the presence ofwhich results in a biological activity of a protein that is the same asthe biological activity resulting from the presence of a naturallyoccurring ligand for the protein, such as, for example, Btk.

As used herein, “ACK” and “Accessible Cysteine Kinase” are synonyms.They mean a kinase with an accessible cysteine residue. ACKS include,but are not limited to, BTK, ITK, Bmx/ETK, TEC, EFGR, HER4, HER4, LCK,BLK, C-src, FGR, Fyn, HCK, Lyn, YES, ABL, Brk, CSK, FER, JAK3, SYK. Insome embodiments, the ACK is HER4.

As used herein, the term “partial agonist” refers to a compound thepresence of which results in a biological activity of a protein that isof the same type as that resulting from the presence of a naturallyoccurring ligand for the protein, but of a lower magnitude.

As used herein, the term “antagonist” refers to a compound, the presenceof which results in a decrease in the magnitude of a biological activityof a protein. In certain embodiments, the presence of an antagonistresults in complete inhibition of a biological activity of a protein,such as, for example, Btk. In certain embodiments, an antagonist is aninhibitor.

As used herein, “amelioration” of the symptoms of a particular disorderby administration of a particular compound or pharmaceutical compositionrefers to any lessening of severity, delay in onset, slowing ofprogression, or shortening of duration, whether permanent or temporary,lasting or transient that can be attributed to or associated withadministration of the compound or composition.

“Bioavailability” refers to the percentage of the weight of compoundsdisclosed herein, such as, compounds of any of Formula (A1-A6), Formula(B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I), or Formula(VII), dosed that is delivered into the general circulation of theanimal or human being studied. The total exposure (AUC_((0-∞))) of adrug when administered intravenously is usually defined as 100%bioavailable (F %). “Oral bioavailability” refers to the extent to whichcompounds disclosed herein, such as, compounds of any of Formula(A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I),or Formula (VII), are absorbed into the general circulation when thepharmaceutical composition is taken orally as compared to intravenousinjection.

The term “biophysical probe,” as used herein, refers to probes whichdetect or monitor structural changes in molecules (includingbiomolecules) in biological systems or in the presence of otherbiomolecules (e.g., ex vivo, in vivo or in vitro). In some embodiments,such molecules include, but are not limited to, proteins and the“biophysical probe” is used to detect or monitor interaction of proteinswith other macromolecules. In other embodiments, examples of biophysicalprobes include, but are not limited to, spin-labels, fluorophores, andphotoactivatable groups.

“Blood plasma concentration” refers to the concentration of compoundsdisclosed herein, such as, compounds of any of Formula (A1-A6), Formula(B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I), or Formula(VII), in the plasma component of blood of an individual. It isunderstood that the plasma concentration of compounds of any of Formula(A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I),or Formula (VII), may vary significantly between subjects, due tovariability with respect to metabolism and/or possible interactions withother therapeutic agents. In accordance with one embodiment disclosedherein, the blood plasma concentration of the compounds of any ofFormula (A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6),Formula (I), or Formula (VII), does vary from subject to subject.Likewise, values such as maximum plasma concentration (C_(max)) or timeto reach maximum plasma concentration (T_(max)), or total area under theplasma concentration time curve (AUC_((0-∞))) may vary from subject tosubject. Due to this variability, the amount necessary to constitute “atherapeutically effective amount” of a compound of any of Formula(A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I),or Formula (VII), is expected to vary from subject to subject.

The term “Bruton's tyrosine kinase,” as used herein, refers to Bruton'styrosine kinase from Homo sapiens, as disclosed in, e.g., U.S. Pat. No.6,326,469 (GenBank Accession No. NP 000052).

The term “Bruton's tyrosine kinase homolog,” as used herein, refers toorthologs of Bruton's tyrosine kinase, e.g., the orthologs from mouse(GenBank Accession No. AAB47246), dog (GenBank Accession No. XP 549139),rat (GenBank Accession No. NP 001007799), chicken (GenBank Accession No.NP 989564), or zebra fish (GenBank Accession No. XP 698117), and fusionproteins of any of the foregoing that exhibit kinase activity towardsone or more substrates of Bruton's tyrosine kinase (e.g. a peptidesubstrate having the amino acid sequence “AVLESEEELYSSARQ” (SEQ ID NO:1)).

The term “HER4”, also known as ERBB4, also known as “V-erb-aerythroblastic leukemia viral oncogene homolog 4” means either (a) thenucleic acid sequence encoding a receptor tyrosine kinase that is amember of the epidermal growth factor receptor subfamily, or (b) theprotein thereof. For the nucleic acid sequence that comprises the humanHER4 gene see GenBank Accession No. NM_001042599. For the amino acidsequence that comprises the human HER4 protein see GenBank Accession No.NP_001036064.

The term “chemiluminescent group,” as used herein, refers to a groupwhich emits light as a result of a chemical reaction without theaddition of heat. By way of example only, luminol(5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants likehydrogen peroxide (H₂O₂) in the presence of a base and a metal catalystto produce an excited state product (3-aminophthalate, 3-APA).

The term “chromophore,” as used herein, refers to a molecule whichabsorbs light of visible wavelengths, UV wavelengths or IR wavelengths.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

In other embodiments, the term “detectable label,” as used herein,refers to a label which is observable using analytical techniquesincluding, but not limited to, fluorescence, chemiluminescence,electron-spin resonance, ultraviolet/visible absorbance spectroscopy,mass spectrometry, nuclear magnetic resonance, magnetic resonance, andelectrochemical methods.

The term “dye,” as used herein, refers to a soluble, coloring substancewhich contains a chromophore.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disorder being treated. The result can be reductionand/or alleviation of the signs, symptoms, or causes of a disease, orany other desired alteration of a biological system. For example, an“effective amount” for therapeutic uses is the amount of the compositionincluding a compound as disclosed herein required to provide aclinically significant decrease in disease symptoms without undueadverse side effects. An appropriate “effective amount” in anyindividual case is optionally determined using techniques, such as adose escalation study. The term “therapeutically effective amount”includes, for example, a prophylactically effective amount. An“effective amount” of a compound disclosed herein is an amount effectiveto achieve a desired pharmacologic effect or therapeutic improvementwithout undue adverse side effects. It is understood that “an effectamount” or “a therapeutically effective amount” can vary from subject tosubject, due to variation in metabolism of the compound of any ofFormula (A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6),Formula (I), or Formula (VII), age, weight, general condition of thesubject, the condition being treated, the severity of the conditionbeing treated, and the judgment of the prescribing physician.

The term “electron dense group,” as used herein, refers to a group whichscatters electrons when irradiated with an electron beam. Such groupsinclude, but are not limited to, ammonium molybdate, bismuth subnitratecadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate,hexamethylene tetramine, 98.5%, indium trichloride anhydrous, lanthanumnitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate,periodic acid, phosphomolybdic acid, phosphotungstic acid, potassiumferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate,silver proteinate (Ag Assay: 8.0-8.5%) “Strong”, silvertetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate,thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranylnitrate, and vanadyl sulfate.

In other embodiments, the term “energy transfer agent,” as used herein,refers to a molecule which either donates or accepts energy from anothermolecule. By way of example only, fluorescence resonance energy transfer(FRET) is a dipole-dipole coupling process by which the excited-stateenergy of a fluorescence donor molecule is non-radiatively transferredto an unexcited acceptor molecule which then fluorescently emits thedonated energy at a longer wavelength.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. By way of example, “enhancing”the effect of therapeutic agents refers to the ability to increase orprolong, either in potency or duration, the effect of therapeutic agentson during treatment of a disorder. An “enhancing-effective amount,” asused herein, refers to an amount adequate to enhance the effect of atherapeutic agent in the treatment of a disorder. When used in anindividual, amounts effective for this use will depend on the severityand course of the disorder, previous therapy, the individual's healthstatus and response to the drugs, and the judgment of the treatingphysician.

The term “fluorophore,” as used herein, refers to a molecule which uponexcitation emits photons and is thereby fluorescent.

The term “homologous cysteine,” as used herein refers to a cysteineresidue found with in a sequence position that is homologous to that ofcysteine 481 of Bruton's tyrosine kinase, as defined herein. Forexample, cysteine 482 is the homologous cysteine of the rat ortholog ofBruton's tyrosine kinase; cysteine 479 is the homologous cysteine of thechicken ortholog; and cysteine 481 is the homologous cysteine in thezebra fish ortholog. In another example, the homologous cysteine of TXK,a Tec kinase family member related to Bruton's tyrosine, is Cys 350.Other examples of kinases having homologous cysteines are shown in FIG.7. See also the sequence alignments of tyrosine kinases (TK) publishedon the world wide web at kinase.com/human/kinome/phylogeny.html.

The term “identical,” as used herein, refers to two or more sequences orsubsequences which are the same. In addition, the term “substantiallyidentical,” as used herein, refers to two or more sequences which have apercentage of sequential units which are the same when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using comparison algorithms or by manualalignment and visual inspection. By way of example only, two or moresequences are “substantially identical” if the sequential units areabout 60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, or about 95% identical over a specified region. Suchpercentages to describe the “percent identity” of two or more sequences.The identity of a sequence can exist over a region that is at leastabout 75-100 sequential units in length, over a region that is about 50sequential units in length, or, where not specified, across the entiresequence. This definition also refers to the complement of a testsequence. By way of example only, two or more polypeptide sequences areidentical when the amino acid residues are the same, while two or morepolypeptide sequences are “substantially identical” if the amino acidresidues are about 60% identical, about 65% identical, about 70%identical, about 75% identical, about 80% identical, about 85%identical, about 90% identical, or about 95% identical over a specifiedregion. The identity can exist over a region that is at least about75-100 amino acids in length, over a region that is about 50 amino acidsin length, or, where not specified, across the entire sequence of apolypeptide sequence. In addition, by way of example only, two or morepolynucleotide sequences are identical when the nucleic acid residuesare the same, while two or more polynucleotide sequences are“substantially identical” if the nucleic acid residues are about 60%identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, or about 95% identical over a specified region. The identitycan exist over a region that is at least about 75-100 nucleic acids inlength, over a region that is about 50 nucleic acids in length, or,where not specified, across the entire sequence of a polynucleotidesequence.

The terms “inhibits”, “inhibiting”, or “inhibitor” of a kinase, as usedherein, refer to inhibition of enzymatic phosphotransferase activity.

The term “irreversible inhibitor,” as used herein, refers to a compoundthat, upon contact with a target protein (e.g., a kinase) causes theformation of a new covalent bond with or within the protein, whereby oneor more of the target protein's biological activities (e.g.,phosphotransferase activity) is diminished or abolished notwithstandingthe subsequent presence or absence of the irreversible inhibitor.

The term “irreversible Btk inhibitor,” as used herein, refers to aninhibitor of Btk that can form a covalent bond with an amino acidresidue of Btk. In one embodiment, the irreversible inhibitor of Btk canform a covalent bond with a Cys residue of Btk; in particularembodiments, the irreversible inhibitor can form a covalent bond with aCys 481 residue (or a homolog thereof) of Btk or a cysteine residue inthe homologous corresponding position of another tyrosine kinase, asshown in FIG. 7.

The term “isolated,” as used herein, refers to separating and removing acomponent of interest from at least some portion of components not ofinterest. Isolated substances can be in either a dry or semi-dry state,or in solution, including but not limited to an aqueous solution. Theisolated component can be in a homogeneous state or the isolatedcomponent can be a part of a pharmaceutical composition that comprisesadditional pharmaceutically acceptable carriers and/or excipients. Byway of example only, nucleic acids or proteins are “isolated” when suchnucleic acids or proteins are free of at least some of the cellularcomponents with which it is associated in the natural state, or that thenucleic acid or protein has been concentrated to a level greater thanthe concentration of its in vivo or in vitro production. Also, by way ofexample, a gene is isolated when separated from open reading frameswhich flank the gene and encode a protein other than the gene ofinterest.

In some embodiments, the term “label,” as used herein, refers to asubstance which is incorporated into a compound and is readily detected,whereby its physical distribution is detected and/or monitored.

The term “linkage,” as used herein to refer to bonds or a chemicalmoiety formed from a chemical reaction between the functional group of alinker and another molecule. In some embodiments, such bonds include,but are not limited to, covalent linkages and non-covalent bonds, whilesuch chemical moieties include, but are not limited to, esters,carbonates, imines, phosphate esters, hydrazones, acetals, orthoesters,peptide linkages, and oligonucleotide linkages. Hydrolytically stablelinkages means that the linkages are substantially stable in water anddo not react with water at useful pH values, including but not limitedto, under physiological conditions for an extended period of time,perhaps even indefinitely. Hydrolytically unstable or degradablelinkages means that the linkages are degradable in water or in aqueoussolutions, including for example, blood. In other embodiments,enzymatically unstable or degradable linkages means that the linkage isdegraded by one or more enzymes. By way of example only, PEG and relatedpolymers include degradable linkages in the polymer backbone or in thelinker group between the polymer backbone and one or more of theterminal functional groups of the polymer molecule. Such degradablelinkages include, but are not limited to, ester linkages formed by thereaction of PEG carboxylic acids or activated PEG carboxylic acids withalcohol groups on a biologically active agent, wherein such ester groupsgenerally hydrolyze under physiological conditions to release thebiologically active agent. Other hydrolytically degradable linkagesinclude but are not limited to carbonate linkages; imine linkagesresulted from reaction of an amine and an aldehyde; phosphate esterlinkages formed by reacting an alcohol with a phosphate group; hydrazonelinkages which are reaction product of a hydrazide and an aldehyde;acetal linkages that are the reaction product of an aldehyde and analcohol; orthoester linkages that are the reaction product of a formateand an alcohol; peptide linkages formed by an amine group, including butnot limited to, at an end of a polymer such as PEG, and a carboxyl groupof a peptide; and oligonucleotide linkages formed by a phosphoramiditegroup, including but not limited to, at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide.

The phrase “measuring the activity of the reporter moiety” (or asimilarly worded phrase) refers to methods for quantifying (in absolute,approximate or relative terms) the reporter moiety in a system understudy. In some embodiments, such methods include any methods thatquantify a reporter moiety that is a dye; a photocrosslinker; acytotoxic compound; a drug; an affinity label; a photoaffinity label; areactive compound; an antibody or antibody fragment; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a ligand; a photoisomerizablemoiety; biotin; a biotin analogue; a moiety incorporating a heavy atom;a chemically cleavable group; a photocleavable group; a redox-activeagent; an isotopically labeled moiety; a biophysical probe; aphosphorescent group; a chemiluminescent group; an electron dense group;a magnetic group; an intercalating group; a chromophore; an energytransfer agent; a biologically active agent; a detectable label; and anycombination of the above.

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes, such as, oxidation reactions) by which aparticular substance is changed by an organism. Thus, enzymes producespecific structural alterations to a compound. For example, cytochromeP450 catalyzes a variety of oxidative and reductive reactions whileuridine diphosphate glucuronyl transferases catalyze the transfer of anactivated glucuronic-acid molecule to aromatic alcohols, aliphaticalcohols, carboxylic acids, amines and free sulfhydryl groups. Furtherinformation on metabolism is obtained from The Pharmacological Basis ofTherapeutics, 9th Edition, McGraw-Hill (1996). Metabolites of thecompounds disclosed herein are optionally identified either byadministration of compounds to a host and analysis of tissue samplesfrom the host, or by incubation of compounds with hepatic cells in vitroand analysis of the resulting compounds. In some embodiments,metabolites of a compound are formed by oxidative processes andcorrespond to the corresponding hydroxy-containing compound. In someembodiments, a compound is metabolized to pharmacologically activemetabolites.

The term “modulate,” as used herein, means to interact with a targeteither directly or indirectly so as to alter the activity of the target,including, by way of example only, to enhance the activity of thetarget, to inhibit the activity of the target, to limit the activity ofthe target, or to extend the activity of the target.

As used herein, the term “modulator” refers to a compound that alters anactivity of a molecule. For example, a modulator can cause an increaseor decrease in the magnitude of a certain activity of a moleculecompared to the magnitude of the activity in the absence of themodulator. In certain embodiments, a modulator is an inhibitor, whichdecreases the magnitude of one or more activities of a molecule. Incertain embodiments, an inhibitor completely prevents one or moreactivities of a molecule. In certain embodiments, a modulator is anactivator, which increases the magnitude of at least one activity of amolecule. In certain embodiments the presence of a modulator results inan activity that does not occur in the absence of the modulator.

The term “moiety incorporating a heavy atom,” as used herein, refers toa group which incorporates an ion of atom which is usually heavier thancarbon. In some embodiments, such ions or atoms include, but are notlimited to, silicon, tungsten, gold, lead, and uranium.

The term “nanoparticle,” as used herein, refers to a particle which hasa particle size between about 500 nm to about 1 nm.

As used herein, the term “pERK” refers to phosphorylated ERK1 and ERK2at Thr202/Tyr 204 as detected by commercially available phospho-specificantibodies (e.g. Cell Signaling Technologies #4377).

The term “photoaffinity label,” as used herein, refers to a label with agroup, which, upon exposure to light, forms a linkage with a moleculefor which the label has an affinity. By way of example only, in someembodiments, such a linkage is covalent or non-covalent.

The term “photocaged moiety,” as used herein, refers to a group which,upon illumination at certain wavelengths, covalently or non-covalentlybinds other ions or molecules.

The term “photoisomerizable moiety,” as used herein, refers to a groupwherein upon illumination with light changes from one isomeric form toanother.

The term “plasma half life,” as used herein refers to half-life in rat,dog or human as determined by measure drug concentration over time inplasma following a single dose and fitting data to standardpharmacokinetic models using software such as WinNonLin to determine thetime at which drug has been 50% eliminated from plasma.

The term “prophylactically effective amount,” as used herein, refersthat amount of a composition applied to an individual which will relieveto some extent one or more of the symptoms of a disease, disorder beingtreated. In such prophylactic applications, such amounts may depend onthe patient's state of health, weight, and the like.

The term “radioactive moiety,” as used herein, refers to a group whosenuclei spontaneously give off nuclear radiation, such as alpha, beta, orgamma particles; wherein, alpha particles are helium nuclei, betaparticles are electrons, and gamma particles are high energy photons.

As used herein, the term “selective binding compound” refers to acompound that selectively binds to any portion of one or more targetproteins.

As used herein, the term “selectively binds” refers to the ability of aselective binding compound to bind to a target protein, such as, forexample, Btk, with greater affinity than it binds to a non-targetprotein. In certain embodiments, specific binding refers to binding to atarget with an affinity that is at least 10, 50, 100, 250, 500, 1000 ormore times greater than the affinity for a non-target.

As used herein, the term “selective modulator” refers to a compound thatselectively modulates a target activity relative to a non-targetactivity. In certain embodiments, specific modulator refers tomodulating a target activity at least 10, 50, 100, 250, 500, 1000 timesmore than a non-target activity.

The term “spin label,” as used herein, refers to molecules which containan atom or a group of atoms exhibiting an unpaired electron spin (i.e. astable paramagnetic group) that in some embodiments are detected byelectron spin resonance spectroscopy and in other embodiments areattached to another molecule. Such spin-label molecules include, but arenot limited to, nitryl radicals and nitroxides, and in some embodimentsare single spin-labels or double spin-labels.

The term “substantially purified,” as used herein, refers to a componentof interest that may be substantially or essentially free of othercomponents which normally accompany or interact with the component ofinterest prior to purification. By way of example only, a component ofinterest may be “substantially purified” when the preparation of thecomponent of interest contains less than about 30%, less than about 25%,less than about 20%, less than about 15%, less than about 10%, less thanabout 5%, less than about 4%, less than about 3%, less than about 2%, orless than about 1% (by dry weight) of contaminating components. Thus, a“substantially purified” component of interest may have a purity levelof about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,about 96%, about 97%, about 98%, about 99% or greater.

The term “individual” as used herein, refers to a mammal which is theobject of treatment, observation or experiment. The term is not to beconstrued as requiring the supervision of a medical practicioner (e.g.,a physician, physician's assistant, nurse, orderly, hospice careworker).

As used herein, the term “target activity” refers to a biologicalactivity capable of being modulated by a selective modulator. Certainexemplary target activities include, but are not limited to, bindingaffinity, signal transduction, enzymatic activity, tumor growth,inflammation or inflammation-related processes, and amelioration of oneor more symptoms associated with a disorder.

As used herein, the term “target protein” refers to a molecule or aportion of a protein capable of being bound by a selective bindingcompound. In certain embodiments, a target protein is Btk.

The terms “treat,” “treating” or “treatment”, as used herein, includealleviating, abating or ameliorating a symptom of a disorder, preventingadditional symptoms, ameliorating or preventing the underlying metaboliccauses of symptoms, inhibiting the disorder, e.g., arresting thedevelopment of the disorder, relieving the disorder, causing regressionof the disorder, relieving a condition caused by the disorder, orstopping the symptoms of the disorder. The terms “treat,” “treating” or“treatment”, include, but are not limited to, prophylactic and/ortherapeutic treatments.

As used herein, the IC₅₀ refers to an amount, concentration or dosage ofa particular test compound that achieves a 50% inhibition of a maximalresponse, such as inhibition of Btk, in an assay that measures suchresponse.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A presents an illustrative table of GI₅₀ concentrations ofCompound 1 that results in 50% decrease in cell proliferation. A varietyof lymphoma cell lines incubated with a range of concentrations ofCompound 1. FIG. 1B presents an illustrative line graph showinginhibition of tumor growth in DLCL2 xenograft models. FIG. 1C presentsan illustrative line graph showing inhibition of tumor growth in DOHH2xenograft models. For in vivo lymphoma xenograft studies, 5E6 DOHH2 orDLCL2 cells in 50% matrigel were implanted subcutaneously in SCID miceand dosed orally with Compound 1 beginning when tumor size reached 100mm2.

FIG. 2 presents an illustrative line graph showing inhibition ofcollagen-induced arthritis in male DBA/1OlaHsd mice. Compound forvehicle was dosed orally once per day starting at day 1. Dexamethasonewas included as a positive control. Paw inflammation was scored from 0-5and averaged across all paws from all animals for each group in thestudy. Compound 1 at 12.5 mg/kg and 50 mg/kg regressed inflammationthrough the end of the study (day 11) while 3.125 mg/kg significantlyreduced the increase in paw inflammation.

FIG. 3 presents an illustrative line graph showing inhibition of diseaseprogression in a mouse MRL/lpr model of lupus. MRL/lpr mice (Jax strain000485) were dosed orally once per day from 8 weeks of age until 20weeks of age and urine protein levels were measured weekly. Compound 1at 3.125 mg/kg, 12.5 mg/kg, and 50 mg/kg significantly reducedproteinuria, indicating amelioration of the progressive autoimmune renalfailure seen in this mouse strain.

FIG. 4 presents an illustrative bar graph showing inhibition of mastcell degranulation in a mouse passive cutaneous anaphylaxis model. 23hours after mice were sensitized with an intradermal injection ofmonoclonal anti-DNP-IgE in the back, they received a single oral dose ofCompound 1 or vehicle. After one hour, animals were challenged with anintravenous injection of DNP-BSA and Evans Blue dye and the area ofextravasation was measured. Increasing doses of Compound 1 significantlydecreased the amount of Evans Blue release, indicating decreased mastcell activation and vascular permeabilization.

FIG. 5 presents an illustrative line graph showing in vivo plasmaconcentrations post-dosing of male jugular vein cannulated rats withCompounds 1, 7, 8, and 12. Blood samples were collected at 0.0833 (5minutes), 0.333 (20 minutes), 1, 3, 6, 9, and 24 hours post-dosing fromorally dosed rats. Compound 1 and Compound 12 have a short half-life invivo. In contrast, Compound 7 and Compound 8 have a significantly longerin vivo half-life. Compounds like 1 and 12 are predicted to haveenhanced kinase selectivity in vivo because inhibition will be sustainedonly for those kinases that are irreversibly inhibited.

FIG. 6A presents an experimental scheme that illustrates a brief invitro exposure of B cells to Compound 1. FIG. 6B presents anillustrative bar graph showing brief exposure to Compound 1 in vitro issufficient to inhibit B cell activation in normal human B cells. B cellswere purified from blood from healthy donors by negative selecting usingthe RosetteSep Human B cell enrichment cocktail. Cells were plated ingrowth media and indicated concentrations of Compound 1 were added.After incubation for 1 hour at 37° C., cells were washed three timesusing an 8-fold dilution in growth media for each wash. Cells were thenstimulated with IgM F(ab′)2 for 18 hours at 37° C., stained withanti-CD69-PE antibody and analyzed by flow cytometry. This protocolmimics the predicted exposure of cells to Compound 1 in vivo anddemonstrates that inhibition of B cells is sustained despite washing outof Compound 1.

FIG. 7 presents illustrative ACKs, including Btk and Btk cysteinehomologs. FIG. 7 discloses SEQ ID NOS: 2-14, respectively, in order ofappearance.

FIG. 8 shows efficacy of HER4 inhibitor Compound 1 in MDA-MB-453 grownas a xenograft in nude mice.

FIG. 9A-FIG. 9D: exemplifies the effects of ibrutinib on relative cellgrowth (FIG. 9A) BT-474 cells—ibrutinib, AVL-292 and PCI-45468; (FIG.9B) SK-BR3 cells—ibrutinib, AVL-292 and PCI-4546; (FIG. 9C) UACC-893cells—ibrutinib; and (FIG. 9D) MDA-MB-453 cells—ibrutinib, AVL-292 andPCI-4546.

FIG. 10A-FIG. 10B: exemplifies the effects of ibrutinib and PCI-24781 onBT-474 cells (FIG. 10A) 3-day treatment: annexin-V; and (FIG. 10B) 3-daytreatment: PI.

FIG. 11: exemplifies the effects of ibrutinib on tumor volume of aMDA-MB-453 mouse xenograft.

FIG. 12: exemplifies gene expression levels of multiple cancer celllines with (+) or without (−) administration of 0.1 uM ibrutinib.

FIG. 13: exemplifies gene expression levels of multiple cancer celllines with (+) or without (−) administration of 0.1 uM ibrutinib.

FIG. 14: is an exemplary comparison of the effects of gefitinib andibrutinib on MDA-MB-453 cells.

FIG. 15: exemplifies the effects of ibrutinib, AVL-292 and PCI-45468 onHER2 and HER activiation and downstream pathways in BT-474 cells.

FIG. 16A-FIG. 16B: exemplifies the effects of ibrutinib, dacomitinib,neratinib, PCI-45466, lapatinib, and gefitinib on relative cell growthof SK-BR3 cells (FIG. 16A) and MDA-MB-453 cells (FIG. 16B).

FIG. 17A-FIG. 17C: exemplifies the effects of ibrutinib, lapatinib,neratinib, dacomitinib, afatinib, and gefitinib on relative cell growthof BT-474 cells (FIG. 17A), SK-BR3 cells (FIG. 17B) and MDA-MB-453 cells(FIG. 17C).

DETAILED DESCRIPTION OF THE INVENTION Solid Tumors

In some embodiments, the compounds and formulations described herein areutilized to treat one or more disorders characterized by the presence ordevelopment of a solid tumor. As used herein, “solid tumors” areneoplasms characterized by an absence of liquid areas. In someembodiments, the solid tumor is benign. In some embodiments, the solidtumor is malignant. In some embodiments, the cancer is characterized bythe presence of one or more solid tumor is a sarcoma, carcinoma, and/orlymphoma.

In some embodiments, the disorder characterized by the presence ordevelopment of one or more solid tumors is a sarcoma. Sarcomas arecancers of the bone, cartilage, fat, muscle, blood vessels, or otherconnective or supportive tissue. Sarcomas include, but are not limitedto, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma,malignant schwannoma, osteosarcoma, soft tissue sarcomas (e.g. alveolarsoft part sarcoma, angiosarcoma, cystosarcoma phylloides,dermatofibrosarcoma, desmoid tumor, epithelioid sarcoma, extraskeletalosteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma,Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma,rhabdomyosarcoma, and synovial sarcoma).

In some embodiments, the disorder characterized by the presence ordevelopment of one or more solid tumors is a lymphoma. Lymphomas aresolid neoplasms that originate in lymphocytes. Hodgkin lymphoma ismarked by the presence of the Reed-Sternberg cell. Non-Hodgkin lymphomasare all lymphomas which are not Hodgkin's lymphoma. Non-Hodgkinlymphomas are further divided into indolent lymphomas and aggressivelymphomas. Non-Hodgkin's lymphomas include, but are not limited to,diffuse large B cell lymphoma; follicular lymphoma, Mucosa-AssociatedLymphatic Tissue lymphoma (MALT), small cell lymphocytic lymphoma,mantle cell lymphoma, Burkitt's lymphoma, mediastinal large B celllymphoma, Waldenström macroglobulinemia, nodal marginal zone B celllymphoma (NMZL), splenic marginal zone lymphoma (SMZL), extranodalmarginal zone B cell lymphoma, intravascular large B cell lymphoma,primary effusion lymphoma, and Lymphomatoid granulomatosis.

In some embodiments, the disorder characterized by the presence ordevelopment of one or more solid tumors is a carcinoma. Carcinomas arecancers that begin in the epithelial cells. By way of non-limitingexample, carcinomas include most breast cancers (e.g. mammary ductalcarcinoma and lobular carcinoma), most pancreatic cancers, most lungcancers (e.g. small cell lung carcinoma, and non-small cell lungcarcinoma), most colon cancers, most kidney cancers, and melanomas. Insome embodiments, the disease is mammary ductal carcinoma, lobularcarcinoma, an adenocarcinoma (e.g. pancreatic cancer and colon cancer),small cell lung carcinoma, non-small cell lung carcinoma, and melanomas.In some embodiments, the disease is breast cancer. In some embodiments,the disease is mammary ductal carcinoma, lobular carcinoma, or acombination thereof. In some embodiments, the breast cancer is ERpositive. In some embodiments, the breast cancer is ER negative. In someembodiments, the breast cancer is progesterone receptor (PgR)-positive.In some embodiments, the breast cancer is PgR-negative. In someembodiments, the disease is pancreatic cancer.

Pancreatic cancer is defined as the presence of malignant tumors of thepancreas. The prognosis for individuals with pancreatic cancer isgenerally regarded as poor. In general only about 10 to 15% of patientsdiagnosed with the disorder will survive for 1 year or more; only about3% live for 5 years or more; and only about 2% live for 10 years ormore. The majority of pancreatic tumors are classified asadenocarcinomas.

Mammary ductal carcinoma is a type of breast cancer. It comes in twoforms. Infiltrating ductal carcinoma (IDC) is an invasive, malignant andabnormal proliferation of neoplastic cells in the breast tissue. Ductalcarcinoma in situ (DCIS), is a noninvasive, possibly malignant neoplasmthat is still confined to the lactiferous ducts, where breast cancermost often originates.

Lobular carcinoma is a neoplasm primarily found in the lobules of agland. It comes in two forms. Lobular carcinoma in situ (LCIS) is acondition caused by neoplastic (but not necessarily cancerous) cells inthe lobules of a breast. Invasive lobular carcinoma (aka infiltratinglobular carcinoma) is a type of breast cancer that begins in the lobulesand then invades surrounding tissues.

The growth and development (e.g. into malignant tumors) of a solid tumorrequires the growth of new blood vessels (i.e. angiogenesis). Thetranscription factor MYC is often overexpressed in cancerous cells. Incertain instances, MYC facilitates angiogenesis in tumors by recruitingmast cells to the tumor. In certain instances, tumor cells will undergohypoxia and cell death if mast cell recruitment is inhibited. In someembodiments, mast cell recruitment is inhibited by the use of a Btkinhibitor. In some embodiments, mast cells are killed (e.g. by necrosisor apoptosis) by the use of a Btk inhibitor.

In some embodiments, the disease is HER2 amplified breast cancer. HER2(Human Epidermal Growth Factor Receptor 2) also known as Neu, ErbB2,CD340 (cluster of differentiation 340) and p185 is an epidermal growthfactor receptor found on cells. The HER2 gene is found on humanchromosome 17. HER2 protein is composed of four plasma membrane-boundreceptor tyrosine kinases. Signaling pathways activated by HER2 proteininclude: mitogen-activated protein kinase (MAPK), phosphoinositide3-kinase (PI3K/Akt), phospholipase C y, protein kinase C (PKC), andsignal transducer and activator of transcription (STAT). As used herein,“HER2-amplified cancer” means a cancer (e.g., breast cancer)characterized by amplification (or, over-expression) of the HER2 gene.Amplification of HER2 gene expression leads to increased membraneexpression of the HER2 protein. Increased expression is associated withincreased dimerization of HER2 proteins with HER3 and HER4. Increaseddimerization leads to increased activation of the HER2 tyrosine kinaseresulting in excessive mitosis and cell replication.

Irreversible Inhibitor Compounds

In the following description of irreversible kinase inhibitor compoundssuitable for use in the methods described herein, definitions ofreferred-to standard chemistry terms may be found in reference works (ifnot otherwise defined herein), including Carey and Sundberg “AdvancedOrganic Chemistry 4th Ed.” Vols. A (2000) and B (2001), Plenum Press,New York. In addition, nucleic acid and amino acid sequences for Btk(e.g., human Btk) are disclosed in, e.g., U.S. Pat. No. 6,326,469.Unless specific definitions are provided, the nomenclature employed inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those known in the art.Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients

The inhibitor compounds described herein are selective for kinaseshaving an accessible cysteine residue (such kinases are also known asAccessible Cysteine Kinases, or ACKs) that is able to form a covalentbond with a Michael acceptor moiety on the inhibitor compound. In someembodiments, the cysteine residue is accessible or becomes accessiblewhen the binding site moiety of the irreversible inhibitor binds to thekinase. That is, the binding site moiety of the irreversible inhibitorbinds to an active site of the ACK and the Michael acceptor moiety ofirreversible inhibitor gains access (in one embodiment the step ofbinding leads to a conformational change in the ACK, thus exposing thecysteine) or is otherwise exposed to the cysteine residue of the ACK; asa result a covalent bond is formed between the “S” of the cysteineresidue and the Michael acceptor of the irreversible inhibitor.Consequently, the binding site moiety of the irreversible inhibitorremains bound or otherwise blocks the active site of the ACK.

In one embodiment, the ACK is Btk, a homolog of Btk or a tyrosine kinasehaving a cysteine residue in an amino acid sequence position that ishomologous to the amino acid sequence position of cysteine 481 in Btk.See, e.g., kinases in FIG. 7. In some embodiments, the ACK is HER4.Inhibitor compounds described herein include a Michael acceptor moiety,a binding site moiety and a linker that links the binding site moietyand the Michael acceptor moiety (and in some embodiments, the structureof the linker provides a conformation, or otherwise directs the Michaelacceptor moiety, so as to improve the selectivity of the irreversibleinhibitor for a particular ACK).

Generally, an irreversible inhibitor compound used in the methodsdescribed herein is identified or characterized in an in vitro assay,e.g., an a cellular biochemical assay or a cellular functional assay.Such assays are useful to determine an in vitro IC₅₀ for an irreversibleinhibitor compound.

For example, a cellular kinase assay is used to determine kinaseactivity after incubation of the kinase in the absence or presence of arange of concentrations of a candidate irreversible inhibitor compound.If the candidate compound is in fact an irreversible inhibitor, kinaseactivity will not be recovered by repeat washing with inhibitor-freemedium. See, e.g., J. B. Smaill, et al. (1999), J. Med. Chem.42(10):1803-1815. Further, covalent complex formation between a Kinaseand a candidate irreversible inhibitor is a useful indicator ofirreversible inhibition of the Kinase that is readily determined by anumber of methods (e.g., mass spectrometry). For example, someirreversible Kinase-inhibitor compounds form a covalent bond with theaforenoted cysteine residue (e.g., via a Michael reaction).

High throughput assays for many a cellular biochemical assays (e.g.,kinase assays) and cellular functional assays (e.g., calcium flux) aredocumented methodologies. In addition, high throughput screening systemsare commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.;Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc.Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.). Thesesystems typically automate entire procedures including all sample andreagent pipetting, liquid dispensing, timed incubations, and finalreadings of the microplate in detector(s) appropriate for the assay.Automated systems thereby allow the identification and characterizationof a large number of irreversible compounds.

In some embodiments, irreversible inhibitor compounds are used for themanufacture of a medicament for treating any of the foregoing conditions(e.g. lymphomas, carcinomas, and/or sarcomas).

In some embodiments, the irreversible inhibitor compound used for themethods described herein inhibits a Kinase activity with an in vitroIC₅₀ of less than 10 μM. (e.g., less than 1 μM, less than 0.5 μM, lessthan 0.4 μM, less than 0.3 μM, less than 0.1, less than 0.08 μM, lessthan 0.06 μM, less than 0.05 μM, less than 0.04 μM, less than 0.03 μM,less than less than 0.02 μM, less than 0.01, less than 0.008 μM, lessthan 0.006 μM, less than 0.005 μM, less than 0.004 μM, less than 0.003μM, less than less than 0.002 μM, less than 0.001, less than 0.00099 μM,less than 0.00098 μM, less than 0.00097 μM, less than 0.00096 μM, lessthan 0.00095 μM, less than 0.00094 μM, less than 0.00093 μM, less than0.00092, or less than 0.00090 μM).

In one embodiment, the irreversible inhibitor compound selectively andirreversibly inhibits an activated form of its target tyrosine kinase(e.g., a phosphorylated form of the tyrosine kinase). For example,activated Btk is transphosphorylated at tyrosine 551. Thus, in theseembodiments the irreversible Btk inhibitor inhibits the target kinase incells only once the target kinase is activated by the signaling events.

Particular Irreversible Inhibitor Compounds for ACKs

Described herein are compounds of any of Formula (A1-A6), Formula(B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I), or Formula(VII). Also described herein are pharmaceutically acceptable salts,pharmaceutically acceptable solvates, pharmaceutically activemetabolites, and pharmaceutically acceptable prodrugs of such compounds.Pharmaceutical compositions that include at least one such compound or apharmaceutically acceptable salt, pharmaceutically acceptable solvate,pharmaceutically active metabolite or pharmaceutically acceptableprodrug of such compound, are provided. In some embodiments, whencompounds disclosed herein contain an oxidizable nitrogen atom, thenitrogen atom is optionally converted to an N-oxide. In certainembodiments, isomers and chemically protected forms of compounds havinga structure represented by any of Formula (A1-A6), Formula (B1-B6),Formula (C1-C6), Formula (D1-D6), Formula (I), or Formula (VII), arealso provided.

In one aspect are compounds (including irreversible inhibitors of ACKs,including Btk and its cysteine homologs) having the structure of Formula(I):

wherein

-   -   L_(a) is CH₂, O, NH or S;    -   Ar is a substituted or unsubstituted aryl, or a substituted or        unsubstituted heteroaryl; and    -   either    -   (a) Y is an optionally substituted group selected from among        alkylene, heteroalkylene, arylene, heteroarylene,        alkylenearylene, alkyleneheteroarylene, alkylenecycloalkylene        and alkyleneheterocycloalkylene;    -   Z is C(═O), NHC(═O), NR^(a)C(═O), NR^(a)S(═O)_(x), where x is 1        or 2, and R^(a) is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl; and either    -   (i) R₇ and R₈ are H;    -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈ alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   (ii) R₆ and R₈ are H;    -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈ alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   (iii) R₇ and R₈ taken together form a bond;    -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   (b) Y is an optionally substituted group selected from        cycloalkylene or heterocycloalkylene;    -   Z is C(═O), NHC(═O), NR^(a)C(═O), NR^(a)S(═O)_(x), where x is 1        or 2, and R^(a) is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl; and either    -   (i) R₇ and R₈ are H;    -   R₆ is substituted or unsubstituted C₁-C₄heteroalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   (ii) R₆ and R₈ are H;    -   R₇ is substituted or unsubstituted C₁-C₄heteroalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   (iii) R₇ and R₈ taken together form a bond;    -   R₆ is substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl,        C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,        substituted or unsubstituted C₃-C₆cycloalkyl, substituted or        unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted        C₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,        C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,        C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); and        pharmaceutically active metabolites, or pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (I). By way of example only, are salts of an aminogroup formed with inorganic acids such as hydrochloric acid, hydrobromicacid, phosphoric acid, sulfuric acid and perchloric acid or with organicacids such as acetic acid, oxalic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid. Further salts include thosein which the counterion is an anion, such as adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.Further salts include those in which the counterion is an cation, suchas sodium, lithium, potassium, calcium, magnesium, ammonium, andquaternary ammonium (substituted with at least one organic moiety)cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (I), including those in which the ester group isselected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (I). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (I). Examples ofN-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

For any and all of the embodiments, substituents can be selected fromamong from a subset of the listed alternatives. For example, in someembodiments, L_(a) is CH₂, O, or NH. In other embodiments, L_(a) is O orNH. In yet other embodiments, L_(a) is O.

In some embodiments, Ar is a substituted or unsubstituted aryl. In yetother embodiments, Ar is a 6-membered aryl. In some other embodiments,Ar is phenyl.

In some embodiments, x is 2. In yet other embodiments, Z is C(═O),OC(═O), NHC(═O), S(═O)_(x), OS(═O)_(x), or NHS(═O)_(x). In some otherembodiments, Z is C(═O), NHC(═O), or NCH₃C(═O).

In some embodiments Y is an optionally substituted group selected fromamong alkylene, heteroalkylene, arylene, heteroarylene, alkylenearylene,alkyleneheteroarylene, and alkyleneheterocycloalkylene.

In some embodiments, Z is C(═O), NHC(═O), NR^(a)C(═O), NR^(a)S(═O)_(w),where x is 1 or 2, and R^(a) is H, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl.

In some embodiments, R₇ and R₈ are H; and R₆ is H, substituted orunsubstituted C₁-C₄alkyl, substituted or unsubstituted C₁-C₄heteroalkyl,C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstitutedC₃-C₆cycloalkyl, substituted or unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedC₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl). In otherembodiments, R₆ and R₈ are H; and R₇ is H, substituted or unsubstitutedC₁-C₄alkyl, substituted or unsubstituted C₁-C₄heteroalkyl,C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstitutedC₃-C₆cycloalkyl, substituted or unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedC₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl). In yet furtherembodiments, R₇ and R₈ taken together form a bond; and R₆ is H,substituted or unsubstituted C₁-C₄alkyl, substituted or unsubstitutedC₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstitutedC₃-C₆cycloalkyl, substituted or unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedC₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).

In some embodiments, Y is an optionally substituted group selected fromcycloalkylene or heterocycloalkylene.

In some embodiments, Z is C(═O), NHC(═O), NR^(a)C(═O), NR^(a)S(═O)_(s),where x is 1 or 2, and R^(a) is H, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl.

In some embodiments, R₇ and R₈ are H; and R₆ is substituted orunsubstituted C₁-C₄heteroalkyl, C₁-C₈hydroxyalkylaminoalkyl,C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstitutedC₃-C₆cycloalkyl, substituted or unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedC₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl). In otherembodiments, R₆ and R₈ are H; and R₇ is substituted or unsubstitutedC₁-C₄heteroalkyl, C₁-C₈hydroxyalkylaminoalkyl,C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstitutedC₃-C₆cycloalkyl, substituted or unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedC₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl). In furtherembodiments, R₇ and R₈ taken together form a bond; and R₆ is substitutedor unsubstituted C₁-C₄alkyl, substituted or unsubstitutedC₁-C₄heteroalkyl, C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstitutedC₃-C₆cycloalkyl, substituted or unsubstituted C₁-C₈alkylC₃-C₆cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedC₂-C₈heterocycloalkyl, substituted or unsubstituted heteroaryl,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers,C₁-C₈alkylamides, or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).

In one aspect are compounds (including irreversible inhibitors of ACKs,including Btk and its cysteine homologs) having the structure of Formula(VII):

-   -   wherein

is a moiety that binds to the active site of a kinase, including atyrosine kinase, further including a Btk kinase cysteine homolog;

-   -   Y is an optionally substituted group selected from among        alkylene, heteroalkylene, arylene, heteroarylene,        heterocycloalkylene, cycloalkylene, alkylenearylene,        alkyleneheteroarylene, alkylenecycloalkylene, and        alkyleneheterocycloalkylene;    -   Z is C(═O), OC(═O), NHC(═O), NCH₃C(═O), C(═S), S(═O)_(x),        OS(═O)_(x), NHS(═O)_(x), where x is 1 or 2;    -   R₇ and R₈ are independently selected from among H, unsubstituted        C₁-C₄ alkyl, substituted C₁-C₄alkyl, unsubstituted        C₁-C₄heteroalkyl, substituted C₁-C₄heteroalkyl, unsubstituted        C₃-C₆cycloalkyl, substituted C₃-C₆cycloalkyl, unsubstituted        C₂-C₆heterocycloalkyl, and substituted C₂-C₆heterocycloalkyl; or    -   R₇ and R₈ taken together form a bond; and    -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl,        C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,        C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted        C₃-C₆cycloalkyl, substituted or unsubstituted aryl, substituted        or unsubstituted C₂-C₈heterocycloalkyl, substituted or        unsubstituted heteroaryl, C₁-C₄alkyl(aryl),        C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl), or        C₁-C₄alkyl(C₂-C₈heterocycloalkyl); and        pharmaceutically active metabolites, or pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (VII). By way of example only, are salts of anamino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid. Further saltsinclude those in which the counterion is an anion, such as adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Furthersalts include those in which the counterion is an cation, such assodium, lithium, potassium, calcium, magnesium, ammonium, and quaternaryammonium (substituted with at least one organic moiety) cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (VII), including those in which the ester group isselected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (VII). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (VII). Examples ofN-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

In some embodiments, x is 2. In yet other embodiments, Z is C(═O),OC(═O), NHC(═O), S(═O)_(x), OS(═O)_(x), or NHS(═O)_(x). In some otherembodiments, Z is C(═O), NHC(═O), or S(═O)₂.

In some embodiments, R₇ and R₈ are independently selected from among H,unsubstituted C₁-C₄ alkyl, substituted C₁-C₄alkyl, unsubstitutedC₁-C₄heteroalkyl, and substituted C₁-C₄heteroalkyl; or R₇ and R₈ takentogether form a bond. In yet other embodiments, each of R₇ and R₈ is H;or R₇ and R₈ taken together form a bond.

In some embodiments, R₆ is H, substituted or unsubstituted C₁-C₄alkyl,substituted or unsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl,C₁-C₈alkylaminoalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl),C₁-C₄alkyl(C₃-C₈cycloalkyl), or C₁-C₄alkyl(C₂-C₈heterocycloalkyl). Insome other embodiments, R₆ is H, substituted or unsubstitutedC₁-C₄alkyl, substituted or unsubstituted C₁-C₄heteroalkyl,C₁-C₆alkoxyalkyl, C₁-C₂alkyl-N(C₁-C₃alkyl)₂, C₁-C₄alkyl(aryl),C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl), orC₁-C₄alkyl(C₂-C₈heterocycloalkyl). In yet other embodiments, R₆ is H,substituted or unsubstituted C₁-C₄alkyl, —CH₂—O—(C₁-C₃alkyl),—CH₂—N(C₁-C₃alkyl)₂, C₁-C₄alkyl(phenyl), or C₁-C₄alkyl(5- or 6-memberedheteroaryl). In yet other embodiments, R₆ is H, substituted orunsubstituted C₁-C₄alkyl, —CH₂—O—(C₁-C₃alkyl), —CH₂—(C₁-C₆alkylamino),C₁-C₄alkyl(phenyl), or C₁-C₄alkyl(5- or 6-membered heteroaryl). In someembodiments, R₆ is H, substituted or unsubstituted C₁-C₄alkyl,—CH₂—O—(C₁-C₃alkyl), —CH₂—N(C₁-C₃alkyl)₂, C₁-C₄alkyl(phenyl), orC₁-C₄alkyl(5- or 6-membered heteroaryl containing 1 or 2 N atoms), orC₁-C₄alkyl(5- or 6-membered heterocycloalkyl containing 1 or 2 N atoms).

In some embodiments, Y is an optionally substituted group selected fromamong alkylene, heteroalkylene, arylene, heteroarylene,heterocycloalkylene, cycloalkylene, alkylenearylene,alkyleneheteroarylene, alkylenecycloalkylene, andalkyleneheterocycloalkylene. In other embodiments, Y is an optionallysubstituted group selected from among C₁-C₆alkylene,C₁-C₆heteroalkylene, 4-, 5-, 6-, or 7-membered cycloalkylene, and 4-,5-, 6-, or 7-membered heterocycloalkylene. In yet other embodiments, Yis an optionally substituted group selected from among C₁-C₆alkylene,C₁-C₆heteroalkylene, 5- or 6-membered cycloalkylene, and 5- or6-membered heterocycloalkylene containing 1 or 2 N atoms. In some otherembodiments, Y is a 5- or 6-membered cycloalkylene, or a 5- or6-membered heterocycloalkylene containing 1 or 2 N atoms. In someembodiments, Y is a 4-, 5-, 6-, or 7-membered cycloalkylene ring; or Yis a 4-, 5-, 6-, or 7-membered heterocycloalkylene ring.

In one aspect are compounds (including irreversible inhibitors of ACKs,including Btk and its cysteine homologs) having the structure of Formula(A1):

wherein

-   -   A is independently selected from N or CR₅;    -   R₁ is H, L₂-(substituted or unsubstituted alkyl),        L₂-(substituted or unsubstituted cycloalkyl), L₂-(substituted or        unsubstituted alkenyl), L₂-(substituted or unsubstituted        cycloalkenyl), L₂-(substituted or unsubstituted heterocycle),        L₂-(substituted or unsubstituted heteroaryl), or L₂-(substituted        or unsubstituted aryl), where L₂ is a bond, O, S, —S(═O),        —S(═O)₂, C(═O), -(substituted or unsubstituted C₁-C₆ alkyl), or        -(substituted or unsubstituted C₂-C₆ alkenyl);    -   R₂ and R₃ are independently selected from H, lower alkyl and        substituted lower alkyl;    -   R₄ is L₃-X-L₄-G, wherein,        -   L₃ is optional, and when present is a bond, or an optionally            substituted group selected from alkyl, heteroalkyl, aryl,            heteroaryl, alkylaryl, alkylheteroaryl, or            alkylheterocycloalkyl;        -   X is optional, and when present is a bond, O, —C(═O), S,            —S(═O), —S(═O)₂, —NH, —NR₉, —NHC(O), —C(O)NH, —NR₉C(O),            —C(O)NR₉, —S(═O)₂NH, —NHS(═O)₂, —S(═O)₂NR₉—, —NR₉S(═O)₂,            —OC(O)NH—, —NHC(O)O—, —OC(O)NR₉—, —NR₉C(O)O—, —CH═NO—,            —ON═CH—, —NR₁₀C(O)NR₁₀—, heteroaryl, aryl,            —NR₁₀C(═NR₁₁)NR₁₀—, —NR₁₀C(═NR₁₁)—, —C(═NR₁₁)NR₁₀—,            —OC(═NR₁₁)—, or —C(═NR₁₁)O—;        -   L₄ is optional, and when present is a bond, substituted or            unsubstituted alkyl, substituted or unsubstituted            cycloalkyl, substituted or unsubstituted alkenyl,            substituted or unsubstituted alkynyl, substituted or            unsubstituted aryl, substituted or unsubstituted heteroaryl,            substituted or unsubstituted heterocycle;        -   or L₃, X and L₄ taken together form a nitrogen containing            heterocyclic ring, or an optionally substituted group            selected from alkyl, heteroalkyl, aryl, heteroaryl,            alkylaryl, alkylheteroaryl, or alkylheterocycloalkyl;            -   G is

-   -   -    where R^(a) is H, substituted or unsubstituted alkyl,            substituted or unsubstituted cycloalkyl; and either R₇ and            R₈ are H;            -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,                substituted or unsubstituted C₁-C₄heteroalkyl,                C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,                C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted                C₃-C₆cycloalkyl, substituted or unsubstituted                C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted                aryl, substituted or unsubstituted                C₂-C₈heterocycloalkyl, substituted or unsubstituted                heteroaryl, C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl),                C₁-C₈alkylethers, C₁-C₈alkylamides, or                C₁-C₄alkyl(C₂-C₈heterocycloalkyl);        -   R₆ and R₈ are H;            -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,                substituted or unsubstituted C₁-C₄heteroalkyl,                C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,                C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted                C₃-C₆cycloalkyl, substituted or unsubstituted                C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted                aryl, substituted or unsubstituted                C₂-C₈heterocycloalkyl, substituted or unsubstituted                heteroaryl, C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl),                C₁-C₈alkylethers, C₁-C₈alkylamides, or                C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or        -   R₇ and R₈ taken together form a bond;            -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,                substituted or unsubstituted C₁-C₄heteroalkyl,                C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,                C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted                C₃-C₆cycloalkyl, substituted or unsubstituted                C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted                aryl, substituted or unsubstituted                C₂-C₈heterocycloalkyl, substituted or unsubstituted                heteroaryl, C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl),                C₁-C₈alkylethers, C₁-C₈alkylamides, or                C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or

    -   R₅ is H, halogen, -L₆-(substituted or unsubstituted C₁-C₃        alkyl), -L₆-(substituted or unsubstituted C₂-C₄ alkenyl),        -L₆-(substituted or unsubstituted heteroaryl), or        -L₆-(substituted or unsubstituted aryl), wherein L₆ is a bond,        O, S, —S(═O), S(═O)₂, NH, C(O), —NHC(O)O, —OC(O)NH, —NHC(O), or        —C(O)NH;

    -   each R₉ is independently selected from among H, substituted or        unsubstituted lower alkyl, and substituted or unsubstituted        lower cycloalkyl;

    -   each R₁₀ is independently H, substituted or unsubstituted lower        alkyl, or substituted or unsubstituted lower cycloalkyl; or

    -   two R₁₀ groups can together form a 5-, 6-, 7-, or 8-membered        heterocyclic ring; or

    -   R₉ and R₁₀ can together form a 5-, 6-, 7-, or 8-membered        heterocyclic ring; or

    -   each R₁₁ is independently selected from H, —S(═O)₂R₈,        —S(═O)₂NH₂, —C(O)R₈, —CN, —NO₂, heteroaryl, or heteroalkyl; and        pharmaceutically active metabolites, pharmaceutically acceptable        solvates, pharmaceutically acceptable salts, or pharmaceutically        acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (A1). By way of example only, are salts of an aminogroup formed with inorganic acids such as hydrochloric acid, hydrobromicacid, phosphoric acid, sulfuric acid and perchloric acid or with organicacids such as acetic acid, oxalic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid. Further salts include thosein which the counterion is an anion, such as adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.Further salts include those in which the counterion is an cation, suchas sodium, lithium, potassium, calcium, magnesium, ammonium, andquaternary ammonium (substituted with at least one organic moiety)cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (A1), including those in which the ester group isselected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (A1). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (A1). Examples ofN-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

In a further or alternative embodiment, the compound of Formula (A1) hasthe following structure of Formula (B1):

wherein:

-   -   Y is an optionally substituted group selected from among        alkylene, heteroalkylene, arylene, heteroarylene,        alkylenearylene, alkyleneheteroarylene, and        alkyleneheterocycloalkylene;    -   each R_(a) is independently H, halogen, —CF₃, —CN, —NO₂, OH,        NH₂, -L_(a)-(substituted or unsubstituted alkyl),        -L_(a)-(substituted or unsubstituted alkenyl),        -L_(a)-(substituted or unsubstituted heteroaryl), or        -L_(a)-(substituted or unsubstituted aryl), wherein L_(a) is a        bond, O, S, —S(═O), —S(═O)₂, NH, C(O), CH₂, —NHC(O)O, —NHC(O),        or —C(O)NH;    -   G is

-   -    where R^(a) is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl; and either    -   R₇ and R₈ are H;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₆ and R₈ are H;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₇ and R₈ taken together form a bond;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring; and pharmaceutically acceptable active        metabolites, pharmaceutically acceptable solvates,        pharmaceutically acceptable salts, or pharmaceutically        acceptable prodrugs thereof.

In further or alternative embodiments, G is selected from among

where R is H, alkyl, alkylhydroxy, heterocycloalkyl, heteroaryl,alkylalkoxy, alkylalkoxyalkyl.

In further or alternative embodiments,

is selected from among

In further or alternative embodiment, the compound of Formula (B1) hasthe following structure of Formula (C1):

-   -   Y is an optionally substituted group selected from among alkyl,        heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, and        alkylheterocycloalkyl;    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring;    -   G is

-   -    where R^(a) is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl; and either    -   R₇ and R₈ are H;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₆ and R₈ are H;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₇ and R₈ taken together form a bond;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); and    -   pharmaceutically acceptable active metabolites, pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In a further or alternative embodiment, the “G” group of any of Formula(A1), Formula (B1), or Formula (C1) is any group that is used to tailorthe physical and biological properties of the molecule. Suchtailoring/modifications are achieved using groups which modulate Michaelacceptor chemical reactivity, acidity, basicity, lipophilicity,solubility and other physical properties of the molecule. The physicaland biological properties modulated by such modifications to G include,by way of example only, enhancing chemical reactivity of Michaelacceptor group, solubility, in vivo absorption, and in vivo metabolism.In addition, in vivo metabolism includes, by way of example only,controlling in vivo PK properties, off-target activities, potentialtoxicities associated with cypP450 interactions, drug-drug interactions,and the like. Further, modifications to G allow for the tailoring of thein vivo efficacy of the compound through the modulation of, by way ofexample, specific and non-specific protein binding to plasma proteinsand lipids and tissue distribution in vivo.

In one aspect are compounds (including irreversible inhibitors of ACKs,including Btk and its cysteine homologs) having the structure of Formula(D1):

wherein

-   -   L_(a) is CH₂, O, NH or S;    -   Ar is an optionally substituted aromatic carbocycle or an        aromatic heterocycle;    -   Y is an optionally substituted group selected from among        alkylene, heteroalkylene, arylene, heteroarylene,        alkylenearylene, alkyleneheteroarylene, and        alkyleneheterocycloalkylene, or combination thereof;    -   Z is C(═O), NHC(═O), NR^(a)C(═O), NR^(a)S(═O)_(x), where x is 1        or 2, and R^(a) is H, substituted or unsubstituted alkyl,        substituted or unsubstituted cycloalkyl; and either    -   R₇ and R₈ are H;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₆ and R₈ are H;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or        -   R₇ and R₈ taken together form a bond;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   or combinations thereof; and        pharmaceutically active metabolites, or pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (D1). By way of example only, are salts of an aminogroup formed with inorganic acids such as hydrochloric acid, hydrobromicacid, phosphoric acid, sulfuric acid and perchloric acid or with organicacids such as acetic acid, oxalic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid. Further salts include thosein which the counterion is an anion, such as adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.Further salts include those in which the counterion is an cation, suchas sodium, lithium, potassium, calcium, magnesium, ammonium, andquaternary ammonium (substituted with at least one organic moiety)cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (D1), including those in which the ester group isselected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (D1). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (D1). Examples ofN-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

In a further or alternative embodiment, L_(a) is O.

In a further or alternative embodiment, Ar is phenyl.

In a further or alternative embodiment, Z is C(═O), NHC(═O), orNCH₃C(═O).

In a further or alternative embodiment, each of R₁, R₂, and R₃ is H.

In one aspect are compounds (including irreversible inhibitors of ACKs,including Btk and its cysteine homologs) having the structure of Formula(D1):

wherein:

-   -   L_(a) is CH₂, O, NH or S;    -   Ar is a substituted or unsubstituted aryl, or a substituted or        unsubstituted heteroaryl;    -   Y is an optionally substituted group selected from among        alkylene, heteroalkylene, arylene, heteroarylene,        alkylenearylene, alkylenehetroarylene, alkylenecycloalkylene and        alkyleneheterocycloalkylene;    -   Z is C(═O), NHC(═O), NR^(a)C(═O), NR^(a)S(═O), where x is 1 or        2, and R^(a) is substituted or unsubstituted alkyl, substituted        or unsubstituted cycloalkyl; and either    -   R₇ and R₈ are H;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl);    -   R₆ and R₈ are H;        -   R₇ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); or    -   R₇ and R₈ taken together form a bond;        -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl,            substituted or unsubstituted C₁-C₄heteroalkyl,            C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,            C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted            C₃-C₆cycloalkyl, substituted or unsubstituted            C₁-C₈alkylC₃-C₆cycloalkyl, substituted or unsubstituted            aryl, substituted or unsubstituted C₂-C₈heterocycloalkyl,            substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),            C₁-C₄alkyl(heteroaryl), C₁-C₈alkylethers, C₁-C₈alkylamides,            or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); and pharmaceutically            active metabolites, or pharmaceutically acceptable solvates,            pharmaceutically acceptable salts, or pharmaceutically            acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (D1). By way of example only, are salts of an aminogroup formed with inorganic acids such as hydrochloric acid, hydrobromicacid, phosphoric acid, sulfuric acid and perchloric acid or with organicacids such as acetic acid, oxalic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid. Further salts include thosein which the counterion is an anion, such as adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.Further salts include those in which the counterion is an cation, suchas sodium, lithium, potassium, calcium, magnesium, ammonium, andquaternary ammonium (substituted with at least one organic moiety)cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (D1), including those in which the ester group isselected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (D1). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (D1). Examples ofN-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

For any and all of the embodiments, substituents can be selected fromamong from a subset of the listed alternatives. For example, in someembodiments, L_(a) is CH₂, O, or NH. In other embodiments, L_(a) is O orNH. In yet other embodiments, L_(a) is O.

In some embodiments, Ar is a substituted or unsubstituted aryl. In yetother embodiments, Ar is a 6-membered aryl. In some other embodiments,Ar is phenyl.

In some embodiments, x is 2. In yet other embodiments, Z is C(═O),OC(═O), NHC(═O), S(═O)_(x), OS(═O)_(x), or NHS(═O)_(x). In some otherembodiments, Z is C(═O), NHC(═O), or S(═O)₂.

In some embodiments, R₇ and R₈ are independently selected from among H,unsubstituted C₁-C₄ alkyl, substituted C₁-C₄alkyl, unsubstitutedC₁-C₄heteroalkyl, and substituted C₁-C₄heteroalkyl; or R₇ and R₈ takentogether form a bond. In yet other embodiments, each of R₇ and R₈ is H;or R₇ and R₈ taken together form a bond.

In some embodiments, R₆ is H, substituted or unsubstituted C₁-C₄alkyl,substituted or unsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl,C₁-C₂alkyl-N(C₁-C₃alkyl)₂, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl), orC₁-C₄alkyl(C₂-C₈heterocycloalkyl). In some other embodiments, R₆ is H,substituted or unsubstituted C₁-C₄alkyl, substituted or unsubstitutedC₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl, C₁-C₂alkyl-N(C₁-C₃alkyl)₂,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl),or C₁-C₄alkyl(C₂-C₈heterocycloalkyl). In yet other embodiments, R₆ is H,substituted or unsubstituted C₁-C₄alkyl, —CH₂—O—(C₁-C₃alkyl),—CH₂—N(C₁-C₃alkyl)₂, C₁-C₄alkyl(phenyl), or C₁-C₄alkyl(5- or 6-memberedheteroaryl). In some embodiments, R₆ is H, substituted or unsubstitutedC₁-C₄alkyl, —CH₂—O—(C₁-C₃alkyl), —CH₂—N(C₁-C₃alkyl)₂,C₁-C₄alkyl(phenyl), or C₁-C₄alkyl(5- or 6-membered heteroaryl containing1 or 2 N atoms), or C₁-C₄alkyl(5- or 6-membered heterocycloalkylcontaining 1 or 2 N atoms).

In some embodiments, Y is an optionally substituted group selected fromamong alkylene, heteroalkylene, cycloalkylene, and heterocycloalkylene.In other embodiments, Y is an optionally substituted group selected fromamong C₁-C₆alkylene, C₁-C₆heteroalkylene, 4-, 5-, 6- or 7-memberedcycloalkylene, and 4-, 5-, 6- or 7-membered heterocycloalkylene. In yetother embodiments, Y is an optionally substituted group selected fromamong C₁-C₆alkylene, C₁-C₆heteroalkylene, 5-, or 6-memberedcycloalkylene, and 5-, or 6-membered heterocycloalkylene containing 1 or2 N atoms. In some other embodiments, Y is a 5-, or 6-memberedcycloalkylene, or a 5-, or 6-membered heterocycloalkylene containing 1or 2 N atoms.

In one aspect are compounds (including irreversible inhibitors of ACKs,including Btk and its cysteine homologs) having the structure of Formula(A2-A6):

wherein

-   -   A is independently selected from N or CR₅;    -   R₁ is H, L₂-(substituted or unsubstituted alkyl),        L₂-(substituted or unsubstituted cycloalkyl), L₂-(substituted or        unsubstituted alkenyl), L₂-(substituted or unsubstituted        cycloalkenyl), L₂-(substituted or unsubstituted heterocycle),        L₂-(substituted or unsubstituted heteroaryl), or L₂-(substituted        or unsubstituted aryl), where L₂ is a bond, O, S, —S(═O),        —S(═O)₂, C(═O), -(substituted or unsubstituted C₁-C₆ alkyl), or        -(substituted or unsubstituted C₂-C₆ alkenyl);    -   R₂ and R₃ are independently selected from H, lower alkyl and        substituted lower alkyl;    -   R₄ is L₃-X-L₄-G, wherein,        -   L₃ is optional, and when present is a bond, optionally            substituted or unsubstituted alkyl, optionally substituted            or unsubstituted cycloalkyl, optionally substituted or            unsubstituted alkenyl, optionally substituted or            unsubstituted alkynyl; X is optional, and when present is a            bond, O, —C(═O), S, —S(═O), —S(═O)₂, —NH, —NR₉, —NHC(O),            —C(O)NH, —NR₉C(O), —C(O)NR₉, —S(═O)₂NH, —NHS(═O)₂,            —S(═O)₂NR₉—, —NR₉S(═O)₂, —OC(O)NH—, —NHC(O)O—, —OC(O)NR₉—,            —NR₉C(O)O—, —CH═NO—, —ON═CH—, —NR₁₀C(O)NR₁₀—, heteroaryl,            aryl, —NR₁₀C(═NR₁₁)NR₁₀—, —NR₁₀C(═NR₁₁)—, —C(═NR₁₁)NR₁₀—,            —OC(═NR₁₁)—, or —C(═NR₁₁)O—; L₄ is optional, and when            present is a bond, substituted or unsubstituted alkyl,            substituted or unsubstituted cycloalkyl, substituted or            unsubstituted alkenyl, substituted or unsubstituted alkynyl,            substituted or unsubstituted aryl, substituted or            unsubstituted heteroaryl, substituted or unsubstituted            heterocycle;    -   or L₃, X and L₄ taken together form a nitrogen containing        heterocyclic ring;    -   G is

-   -    wherein,        -   R₆, R₇ and R₈ are independently selected from among H, lower            alkyl or substituted lower alkyl, lower heteroalkyl or            substituted lower heteroalkyl, substituted or unsubstituted            lower cycloalkyl, and substituted or unsubstituted lower            heterocycloalkyl;    -   R₅ is H, halogen, -L₆-(substituted or unsubstituted C₁-C₃        alkyl), -L₆-(substituted or unsubstituted C₂-C₄ alkenyl),        -L₆-(substituted or unsubstituted heteroaryl), or        -L₆-(substituted or unsubstituted aryl), wherein L₆ is a bond,        O, S, —S(═O), S(═O)₂, NH, C(O), —NHC(O)O, —OC(O)NH, —NHC(O), or        —C(O)NH;    -   each R₉ is independently selected from among H, substituted or        unsubstituted lower alkyl, and substituted or unsubstituted        lower cycloalkyl;    -   each R₁₀ is independently H, substituted or unsubstituted lower        alkyl, or substituted or unsubstituted lower cycloalkyl; or        -   two R₁₀ groups can together form a 5-, 6-, 7-, or 8-membered            heterocyclic ring; or    -   R₉ and R₁₀ can together form a 5-, 6-, 7-, or 8-membered        heterocyclic ring; or each R₁₁ is independently selected from H,        —S(═O)₂R₈, —S(═O)₂NH₂, —C(O)R₈, —CN, —NO₂, heteroaryl, or        heteroalkyl; and        pharmaceutically active metabolites, or pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (A2-A6). By way of example only, are salts of anamino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid. Further saltsinclude those in which the counterion is an anion, such as adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Furthersalts include those in which the counterion is an cation, such assodium, lithium, potassium, calcium, magnesium, ammonium, and quaternaryammonium (substituted with at least one organic moiety) cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (A2-A6), including those in which the ester groupis selected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (A2-A6). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (A2-A6). Examplesof N-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

In a further or alternative embodiment, the compound of Formula (A2-A6)has the following structure of Formula (B2-B6):

wherein:

-   -   Y is alkylene or substituted alkylene, or a 4-, 5-, or        6-membered cycloalkylene ring;    -   each R_(a) is independently H, halogen, —CF₃, —CN, —NO₂, OH,        NH₂, -L_(a)-(substituted or unsubstituted alkyl),        -L_(a)-(substituted or unsubstituted alkenyl),        -L_(a)-(substituted or unsubstituted heteroaryl), or        -L_(a)-(substituted or unsubstituted aryl), wherein L_(a) is a        bond, O, S, —S(═O), —S(═O)₂, NH, C(O), CH₂, —NHC(O)O, —NHC(O),        or —C(O)NH;    -   G is

wherein,

-   -   R₆, R₇ and R₈ are independently selected from among H, lower        alkyl or substituted lower alkyl, lower heteroalkyl or        substituted lower heteroalkyl, substituted or unsubstituted        lower cycloalkyl, and substituted or unsubstituted lower        heterocycloalkyl;    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring; and        pharmaceutically acceptable active metabolites, pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In further or alternative embodiments, G is selected from among

In further or alternative embodiments,

is selected from among

In further or alternative embodiment, the compound of Formula (B2-B6)has the following structure of Formula (C2-C6):

-   -   Y is alkylene or substituted alkylene, or a 4-, 5-, or        6-membered cycloalkylene ring;    -   R₁₂ is H or lower alkyl; or    -   Y and R₁₂ taken together form a 4-, 5-, or 6-membered        heterocyclic ring;    -   G is

wherein,

-   -   R₆, R₇ and R₈ are independently selected from among H, lower        alkyl or substituted lower alkyl, lower heteroalkyl or        substituted lower heteroalkyl, substituted or unsubstituted        lower cycloalkyl, and substituted or unsubstituted lower        heterocycloalkyl; and        pharmaceutically acceptable active metabolites, pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In a further or alternative embodiment, the “G” group of any of Formula(A2-A6), Formula (B2-B6), or Formula (C2-C6) is any group that is usedto tailor the physical and biological properties of the molecule. Suchtailoring/modifications are achieved using groups which modulate Michaelacceptor chemical reactivity, acidity, basicity, lipophilicity,solubility and other physical properties of the molecule. The physicaland biological properties modulated by such modifications to G include,by way of example only, enhancing chemical reactivity of Michaelacceptor group, solubility, in vivo absorption, and in vivo metabolism.In addition, in vivo metabolism includes, by way of example only,controlling in vivo PK properties, off-target activities, potentialtoxicities associated with cypP450 interactions, drug-drug interactions,and the like. Further, modifications to G allow for the tailoring of thein vivo efficacy of the compound through the modulation of, by way ofexample, specific and non-specific protein binding to plasma proteinsand lipids and tissue distribution in vivo.

In one aspect are compounds (including irreversible inhibitors of ACKs,including Btk and its cysteine homologs) having the structure of Formula(D2-D6):

wherein

-   -   L_(a) is CH₂, O, NH or S;    -   Ar is an optionally substituted aromatic carbocycle or an        aromatic heterocycle;    -   Y is an optionally substituted alkylene, heteroalkylene,        carbocycloalkylene, heterocycloalkylene, or combination thereof;    -   Z is C(O), OC(O), NHC(O), C(S), S(O)_(x), OS(O)_(x), NHS(O)_(x),        where x is 1 or 2; and    -   R₆, R₇, and R₈ are independently selected from H, alkyl,        heteroalkyl, carbocycle, heterocycle, or combinations thereof;        and        pharmaceutically active metabolites, or pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (D2-D6). By way of example only, are salts of anamino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid. Further saltsinclude those in which the counterion is an anion, such as adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Furthersalts include those in which the counterion is an cation, such assodium, lithium, potassium, calcium, magnesium, ammonium, and quaternaryammonium (substituted with at least one organic moiety) cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (D2-D6), including those in which the ester groupis selected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (D2-D6). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (D2-D6). Examplesof N-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

In a further or alternative embodiment, L_(a) is O.

In a further or alternative embodiment, Ar is phenyl.

In a further or alternative embodiment, Z is C(O).

In a further or alternative embodiment, each of R₁, R₂, and R₃ is H.

In one aspect are compounds (including irreversible inhibitors of ACKs,including Btk and its cysteine homologs) having the structure of Formula(D2-D6):

wherein:

-   -   L_(a) is CH₂, O, NH or S;    -   Ar is a substituted or unsubstituted aryl, or a substituted or        unsubstituted heteroaryl;    -   Y is an optionally substituted group selected from among        alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene,        arylene, and heteroarylene;    -   Z is C(═O), OC(═O), NHC(═O), C(═S), S(═O)_(x), OS(═O)_(x),        NHS(═O)_(x), where x is 1 or 2;    -   R₇ and R₈ are independently selected from among H, unsubstituted        C₁-C₄alkyl, substituted C₁-C₄alkyl, unsubstituted        C₁-C₄heteroalkyl, substituted C₁-C₄heteroalkyl, unsubstituted        C₃-C₆cycloalkyl, substituted C₃-C₆cycloalkyl, unsubstituted        C₂-C₆heterocycloalkyl, and substituted C₂-C₆heterocycloalkyl; or    -   R₇ and R₈ taken together form a bond;    -   R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or        unsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl,        C₁-C₈alkylaminoalkyl, substituted or unsubstituted        C₃-C₆cycloalkyl, substituted or unsubstituted aryl, substituted        or unsubstituted C₂-C₈heterocycloalkyl, substituted or        unsubstituted heteroaryl, C₁-C₄alkyl(aryl),        C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl), or        C₁-C₄alkyl(C₂-C₈heterocycloalkyl); and        pharmaceutically active metabolites, or pharmaceutically        acceptable solvates, pharmaceutically acceptable salts, or        pharmaceutically acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts ofcompounds of Formula (D2-D6). By way of example only, are salts of anamino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid. Further saltsinclude those in which the counterion is an anion, such as adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Furthersalts include those in which the counterion is an cation, such assodium, lithium, potassium, calcium, magnesium, ammonium, and quaternaryammonium (substituted with at least one organic moiety) cations.

In another embodiment are pharmaceutically acceptable esters ofcompounds of Formula (D2-D6), including those in which the ester groupis selected from a formate, acetate, propionate, butyrate, acrylate andethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates ofcompounds of Formula (D2-D6). In another embodiment are pharmaceuticallyacceptable N-acyl derivatives of compounds of Formula (D2-D6).

For any and all of the embodiments, substituents can be selected fromamong from a subset of the listed alternatives. For example, in someembodiments, L_(a) is CH₂, O, or NH. In other embodiments, L_(a) is O orNH. In yet other embodiments, L_(a) is O.

In some embodiments, Ar is a substituted or unsubstituted aryl. In yetother embodiments, Ar is a 6-membered aryl. In some other embodiments,Ar is phenyl.

In some embodiments, x is 2. In yet other embodiments, Z is C(═O),OC(═O), NHC(═O), S(═O)_(x), OS(═O)_(x), or NHS(═O)_(x). In some otherembodiments, Z is C(═O), NHC(═O), or S(═O)₂.

In some embodiments, R₇ and R₈ are independently selected from among H,unsubstituted C₁-C₄ alkyl, substituted C₁-C₄alkyl, unsubstitutedC₁-C₄heteroalkyl, and substituted C₁-C₄heteroalkyl; or R₇ and R₈ takentogether form a bond. In yet other embodiments, each of R₇ and R₈ is H;or R₇ and R₈ taken together form a bond.

In some embodiments, R₆ is H, substituted or unsubstituted C₁-C₄alkyl,substituted or unsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl,C₁-C₂alkyl-N(C₁-C₃alkyl)₂, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, C₁-C₄alkyl(aryl),C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl), orC₁-C₄alkyl(C₂-C₈heterocycloalkyl). In some other embodiments, R₆ is H,substituted or unsubstituted C₁-C₄alkyl, substituted or unsubstitutedC₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl, C₁-C₂alkyl-N(C₁-C₃alkyl)₂,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl),or C₁-C₄alkyl(C₂-C₈heterocycloalkyl). In yet other embodiments, R₆ is H,substituted or unsubstituted C₁-C₄alkyl, —CH₂—O—(C₁-C₃alkyl),—CH₂—N(C₁-C₃alkyl)₂, C₁-C₄alkyl(phenyl), or C₁-C₄alkyl(5- or 6-memberedheteroaryl). In some embodiments, R₆ is H, substituted or unsubstitutedC₁-C₄alkyl, —CH₂—O—(C₁-C₃alkyl), —CH₂—N(C₁-C₃alkyl)₂,C₁-C₄alkyl(phenyl), or C₁-C₄alkyl(5- or 6-membered heteroaryl containing1 or 2 N atoms), or C₁-C₄alkyl(5- or 6-membered heterocycloalkylcontaining 1 or 2 N atoms).

In some embodiments, Y is an optionally substituted group selected fromamong alkylene, heteroalkylene, cycloalkylene, and heterocycloalkylene.In other embodiments, Y is an optionally substituted group selected fromamong C₁-C₆alkylene, C₁-C₆heteroalkylene, 4-, 5-, 6- or 7-memberedcycloalkylene, and 4-, 5-, 6- or 7-membered heterocycloalkylene. In yetother embodiments, Y is an optionally substituted group selected fromamong C₁-C₆alkylene, C₁-C₆heteroalkylene, 5-, or 6-memberedcycloalkylene, and 5-, or 6-membered heterocycloalkylene containing 1 or2 N atoms. In some other embodiments, Y is a 5-, or 6-memberedcycloalkylene, or a 5-, or 6-membered heterocycloalkylene containing 1or 2 N atoms.

Any combination of the groups described above for the various variablesis contemplated herein.

In further aspects are compounds (including irreversible inhibitors ofACKs, including Btk and its cysteine homologs) having the structure ofcompounds of Formula (A1-A6), Formula (B1-B6), Formula (C1-C6), Formula(D1-D6), including, but are not limited to, compounds selected from thegroup consisting of:

In one aspect are compounds (including irreversible inhibitors of ACKs,including Btk and its cysteine homologs) selected from among:(E)-4-(N-(2-hydroxyethyl)-N-methylamino)-1-(3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)but-2-en-1-one(Compound 3);(E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-(1H-imidazol-4-yl)prop-2-en-1-one(Compound 4);(E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-morpholinobut-2-en-1-one(Compound 5);(E)-1-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-(dimethylamino)but-2-en-1-one(Compound 7);(E)-N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)-4-(dimethylamino)but-2-enamide(Compound 8);N-((1r,4r)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)acrylamide(Compound 10);(E)-1-((R)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrolidin-1-yl)-4-(dimethylamino)but-2-en-1-one(Compound 11);(E)-1-((S)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrolidin-1-yl)-4-(dimethylamino)but-2-en-1-one(Compound 12);1-((R)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)prop-2-en-1-one(Compound 13);1-((S)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)prop-2-en-1-one(Compound 14);1((R)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)but-2-yn-1-one(Compound 15);1-((S)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)but-2-yn-1-one(Compound 16);1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)but-2-yn-1-one(Compound 17);(E)-N-((1,r,4r)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl-4-(dimethylamino)but-2-enamide(Compound 18);N-(2-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-methylacrylamide(Compound 19);(E)-1-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-morpholinobut-2-en-1-one(Compound 20); (E)-1-((Samino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)-4-morpholinobut-2-en-1-one(Compound 21);N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)but-2-ynamide(Compound 22);N-(2-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)acrylamide(Compound 23);(E)-1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-morpholinobut-2-en-1-one(Compound 24);(E)-N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)-4-morpholinobut-2-enamide(Compound 25).

The compounds of any of Formula (I), Formula (VII), Formula (A1-A6),Formula (B1-B6), Formula (C1-C6), or Formula (D1-D6) irreversiblyinhibit Btk and are optionally used to treat patients suffering fromBruton's tyrosine kinase-dependent or Bruton's tyrosine kinase mediatedconditions or diseases, including, but not limited to, conditions ordiseases characterized by the presence or development of one or moresolid tumors.

Preparation of Compounds

Compounds of any of Formula (A1-A6), Formula (B1-B6), Formula (C1-C6),Formula (D1-D6), Formula (I), or Formula (VII) are optionallysynthesized using standard synthetic techniques or using such methodsknown in combination with methods described herein. In additions,solvents, temperatures and other reaction conditions are presentedherein for illustration only, and not to limit the scope of the methodsand compositions described herein. As a further guide the followingsynthetic methods may also be utilized.

The reactions are optionally employed in a linear sequence to providethe compounds described herein or used to synthesize fragments which aresubsequently joined by the methods described herein and/or documentedelsewhere.

Formation of Covalent Linkages by Reaction of an Electrophile with aNucleophile

The compounds described herein can be modified using variouselectrophiles or nucleophiles to form new functional groups orsubstituents. Table 1 entitled “Examples of Covalent Linkages andPrecursors Thereof” lists selected examples of covalent linkages andprecursor functional groups which yield and can be used as guidancetoward the variety of electrophiles and nucleophiles combinationsavailable. Precursor functional groups are shown as electrophilic groupsand nucleophilic groups.

TABLE 1 Examples of Covalent Linkages and Precursors Thereof CovalentLinkage Product Electrophile Nucleophile Carboxamides Activated estersamines/anilines Carboxamides acyl azides amines/anilines Carboxamidesacyl halides amines/anilines Esters acyl halides alcohols/phenols Estersacyl nitriles alcohols/phenols Carboxamides acyl nitrilesamines/anilines Imines Aldehydes amines/anilines Hydrazones aldehydes orketones Hydrazines Oximes aldehydes or ketones Hydroxylamines Alkylamines alkyl halides amines/anilines Esters alkyl halides carboxylicacids Thioethers alkyl halides Thiols Ethers alkyl halidesalcohols/phenols Thioethers alkyl sulfonates Thiols Esters alkylsulfonates carboxylic acids Ethers alkyl sulfonates alcohols/phenolsEsters Anhydrides alcohols/phenols Carboxamides Anhydridesamines/anilines Thiophenols aryl halides Thiols Aryl amines aryl halidesAmines Thioethers Azindines Thiols Boronate esters Boronates GlycolsCarboxamides carboxylic acids amines/anilines Esters carboxylic acidsAlcohols hydrazines Hydrazides carboxylic acids N-acylureas orAnhydrides carbodiimides carboxylic acids Esters diazoalkanes carboxylicacids Thioethers Epoxides Thiols Thioethers haloacetamides ThiolsAmmotriazines halotriazines amines/anilines Triazinyl ethershalotriazines alcohols/phenols Amidines imido esters amines/anilinesUreas Isocyanates amines/anilines Urethanes Isocyanates alcohols/phenolsThioureas isothiocyanates amines/anilines Thioethers Maleimides ThiolsPhosphite esters phosphoramidites Alcohols Silyl ethers silyl halidesAlcohols Alkyl amines sulfonate esters amines/anilines Thioetherssulfonate esters Thiols Esters sulfonate esters carboxylic acids Etherssulfonate esters Alcohols Sulfonamides sulfonyl halides amines/anilinesSulfonate esters sulfonyl halides phenols/alcohols Alkyl thiolα,β-unsaturated ester thiols Alkyl ethers α,β-unsaturated ester alcoholsAlkyl amines α,β-unsaturated ester amines Alkyl thiol Vinyl sulfonethiols Alkyl ethers Vinyl sulfone alcohols Alkyl amines Vinyl sulfoneamines Vinyl sulfide Propargyl amide thiol

Use of Protecting Groups

In the reactions described, it may be necessary to protect reactivefunctional groups, for example hydroxy, amino, imino, thio or carboxygroups, where these are desired in the final product, to avoid theirunwanted participation in the reactions. Protecting groups are used toblock some or all reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved. In one embodiment, each protective group be removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions fulfill the requirement of differentialremoval. Protective groups can be removed by acid, base, andhydrogenolysis. Groups such as trityl, dimethoxytrityl, acetal andt-butyldimethylsilyl are acid labile and may be used to protect carboxyand hydroxy reactive moieties in the presence of amino groups protectedwith Cbz groups, which are removable by hydrogenolysis, and Fmoc groups,which are base labile. Carboxylic acid and hydroxy reactive moieties maybe blocked with base labile groups such as, but not limited to, methyl,ethyl, and acetyl in the presence of amines blocked with acid labilegroups such as t-butyl carbamate or with carbamates that are both acidand base stable but hydrolytically removable.

Carboxylic acid and hydroxy reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids may be blockedwith base labile groups such as Fmoc. Carboxylic acid reactive moietiesmay be protected by conversion to simple ester compounds as exemplifiedherein, or they may be blocked with oxidatively-removable protectivegroups such as 2,4-dimethoxybenzyl, while co-existing amino groups maybe blocked with fluoride labile silyl carbamates.

Allyl blocking groups are useful in then presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with a Pd⁰-catalyzedreaction in the presence of acid labile t-butyl carbamate or base-labileacetate amine protecting groups. Yet another form of protecting group isa resin to which a compound or intermediate may be attached. As long asthe residue is attached to the resin, that functional group is blockedand cannot react. Once released from the resin, the functional group isavailable to react.

Typically blocking/protecting groups may be selected from:

Other protecting groups, plus a detailed description of techniquesapplicable to the creation of protecting groups and their removal aredescribed in Greene and Wuts, Protective Groups in Organic Synthesis,3rd Ed., John Wiley & Sons, New York, N.Y., 1999, and Kocienski,Protective Groups, Thieme Verlag, New York, N.Y., 1994, which areincorporated herein by reference for such disclosure.

Synthesis of Compounds

In certain embodiments, provided herein are methods of making andmethods of using tyrosine kinase inhibitor compounds described herein.In certain embodiments, compounds described herein can be synthesizedusing the following synthetic schemes. Compounds may be synthesizedusing methodologies analogous to those described below by the use ofappropriate alternative starting materials.

Described herein are compounds that inhibit the activity of tyrosinekinase(s), such as Btk, and processes for their preparation. Alsodescribed herein are pharmaceutically acceptable salts, pharmaceuticallyacceptable solvates, pharmaceutically active metabolites andpharmaceutically acceptable prodrugs of such compounds. Pharmaceuticalcompositions that include at least one such compound or apharmaceutically acceptable salt, pharmaceutically acceptable solvate,pharmaceutically active metabolite or pharmaceutically acceptableprodrug of such compound, are provided.

The starting material used for the synthesis of the compounds describedherein is either synthesized or obtained from commercial sources, suchas, but not limited to, Aldrich Chemical Co. (Milwaukee, Wis.), Bachem(Torrance, Calif.), or Sigma Chemical Co. (St. Louis, Mo.). Thecompounds described herein, and other related compounds having differentsubstituents are optionally synthesized using techniques and materials,such as described, for example, in March, ADVANCED ORGANIC CHEMISTRY4^(th) Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY4^(th) Ed., Vols. A and B (Plenum 2000, 2001); Green and Wuts,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 3^(rd) Ed., (Wiley 1999); Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); and Larock's ComprehensiveOrganic Transformations (VCH Publishers Inc., 1989). Other methods forthe synthesis of compounds described herein may be found inInternational Patent Publication No. WO 01/01982901, Arnold et al.Bioorganic & Medicinal Chemistry Letters 10 (2000) 2167-2170; Burchat etal. Bioorganic & Medicinal Chemistry Letters 12 (2002) 1687-1690. As aguide the following synthetic methods may be utilized.

The products of the reactions are optionally isolated and purified, ifdesired, using conventional techniques, including, but not limited to,filtration, distillation, crystallization, chromatography and the like.Such materials are optionally characterized using conventional means,including physical constants and spectral data.

Compounds described herein are optionally prepared using the syntheticmethods described herein as a single isomer or a mixture of isomers.

A non-limiting example of a synthetic approach towards the preparationof compounds of any of Formula (A1-A6), Formula (B1-B6), Formula(C1-C6), Formula (D1-D6), Formula (I), or Formula (VII) is shown inScheme I.

Halogenation of commercially available1H-pyrazolo[3,4-d]pyrimidin-4-amine provides an entry into the synthesisof compounds of Formula (A1-A6), (B1-B6), (C1-C6) and/or (D1-D6). In oneembodiment, 1H-pyrazolo[3,4-d]pyrimidin-4-amine is treated withN-iodosuccinamide to give 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine.Metal catalyzed cross coupling reactions are then carried out on3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine. In one embodiment, palladiummediated cross-coupling of a suitably substituted phenyl boronic acidunder basic conditions constructs intermediate 2. Intermediate 2 iscoupled with N-Boc-3-hydroxypiperidine (as non-limiting example) viaMitsunobu reaction to give the Boc (tert-butyloxycarbonyl) protectedintermediate 3. After deprotection with acid, coupling with, but notlimited to, an acid chloride, such as, but not limited to, acryloylchloride, completes the synthesis to give Compound 13.

A non-limiting example of a synthetic approach towards the preparationof compounds containing the imidazotriazine moiety,

is shown in Scheme II.

A non-limiting example of a synthetic approach towards the preparationof compounds containing any imidazopyrazine moiety,

is shown in Scheme III.

A non-limiting example of a synthetic approach towards the preparationof compounds containing the pyrrolopyrimidine moiety,

is shown in Scheme IV.

A non-limiting example of a synthetic approach towards the preparationof compounds containing the Azaindole moiety,

is shown in Scheme V.

A non-limiting example of a synthetic approach towards the preparationof compounds containing the pyrrolopyrimidine moiety,

is shown in Scheme VI.

Using the synthetic methods described herein, tyrosine kinase inhibitorsas disclosed herein are obtained in good yields and purity. Thecompounds prepared by the methods disclosed herein are purified byconventional means, such as, for example, filtration, recrystallization,chromatography, distillation, and combinations thereof.

Any combination of the groups described above for the various variablesis contemplated herein.

Further Forms of Compounds

Compounds disclosed herein have a structure of any of Formula (A1-A6),Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I), orFormula (VII). It is understood that when reference is made to compoundsdescribed herein, it is meant to include compounds of any of Formula(A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I),or Formula (VII), as well as to all of the specific compounds that fallwithin the scope of these generic formulae, unless otherwise indicated.

The compounds described herein may possess one or more stereocenters andeach center may exist in the R or S configuration. The compoundspresented herein include all diastereomeric, enantiomeric, and epimericforms as well as the appropriate mixtures thereof. Stereoisomers may beobtained, if desired, by methods such as, for example, the separation ofstereoisomers by chiral chromatographic columns.

Diasteromeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods known, for example, by chromatography and/or fractionalcrystallization. In one embodiment, enantiomers can be separated bychiral chromatographic columns. In other embodiments, enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,alcohol), separating the diastereomers and converting (e.g.,hydrolyzing) the individual diastereomers to the corresponding pureenantiomers. All such isomers, including diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein.

The methods and formulations described herein include the use ofN-oxides, crystalline forms (also known as polymorphs), orpharmaceutically acceptable salts of compounds described herein, as wellas active metabolites of these compounds having the same type ofactivity. In some situations, compounds exist as tautomers. Alltautomers are included within the scope of the compounds presentedherein. In addition, the compounds described herein can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. The solvated forms of thecompounds presented herein are also considered to be disclosed herein.

Compounds of any of Formula (A1-A6), Formula (B1-B6), Formula (C1-C6),Formula (D1-D6), Formula (I), or Formula (VII) in unoxidized form can beprepared from N-oxides of compounds of any of Formula (A1-A6), Formula(B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I), or Formula (VII)by treating with a reducing agent, such as, but not limited to, sulfur,sulfur dioxide, triphenyl phosphine, lithium borohydride, sodiumborohydride, phosphorus trichloride, tribromide, or the like in asuitable inert organic solvent, such as, but not limited to,acetonitrile, ethanol, aqueous dioxane, or the like at 0 to 80° C.

In some embodiments, compounds described herein are prepared asprodrugs. A “prodrug” refers to an agent that is converted into theparent drug in vivo. Prodrugs are often useful because, in somesituations, they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent is not. The prodrug may also have improved solubility inpharmaceutical compositions over the parent drug. An example, withoutlimitation, of a prodrug is a compound described herein, which isadministered as an ester (the “prodrug”) to facilitate transmittalacross a cell membrane where water solubility is detrimental to mobilitybut which then is metabolically hydrolyzed to the carboxylic acid, theactive entity, once inside the cell where water-solubility isbeneficial. A further example of a prodrug is a short peptide(polyaminoacid) bonded to an acid group where the peptide is metabolizedto reveal the active moiety. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically active form of the compound. Incertain embodiments, a prodrug is enzymatically metabolized by one ormore steps or processes to the biologically, pharmaceutically ortherapeutically active form of the compound. To produce a prodrug, apharmaceutically active compound is modified such that the activecompound will be regenerated upon in vivo administration. The prodrugcan be designed to alter the metabolic stability or the transportcharacteristics of a drug, to mask side effects or toxicity, to improvethe flavor of a drug or to alter other characteristics or properties ofa drug. By virtue of knowledge of pharmacodynamic processes and drugmetabolism in vivo, once a pharmaceutically active compound is known,prodrugs of compounds can be designed (if desired) (for examples of thisprocedure applied to other compounds, see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392; Silverman (1992), The Organic Chemistry of DrugDesign and Drug Action, Academic Press, Inc., San Diego, pages 352-401,Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters,Vol. 4, p. 1985).

Prodrug forms of the herein described compounds, wherein the prodrug ismetabolized in vivo to produce a derivative as set forth herein areincluded within the scope of the claims. In some cases, some of thecompounds herein-described are prodrugs for another derivative or activecompound.

Prodrugs are often useful because, in some situations, they are easierto administer than the parent drug. They are, for instance, bioavailableby oral administration whereas the parent is not. The prodrug optionallyhas improved solubility in pharmaceutical compositions over the parentdrug. Prodrugs may be designed as reversible drug derivatives, for useas modifiers to enhance drug transport to site-specific tissues. In someembodiments, the design of a prodrug increases the effective watersolubility. See, e.g., Fedorak et al., Am. J. Physiol., 269:G210-218(1995); McLoed et al., Gastroenterol, 106:405-413 (1994); Hochhaus etal., Biomed. Chrom., 6:283-286 (1992); J. Larsen and H. Bundgaard, Int.J. Pharmaceutics, 37, 87 (1987); J. Larsen et al., Int. J.Pharmaceutics, 47, 103 (1988); Sinkula et al., J. Pharm. Sci.,64:181-210 (1975); T. Higuchi and V. Stella, Pro-drugs as Novel DeliverySystems, Vol. 14 of the A.C.S. Symposium Series; and Edward B. Roche,Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, all incorporated by reference forsuch disclosure.

Sites on the aromatic ring portion of compounds of any of Formula(A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I),or Formula (VII) can be susceptible to various metabolic reactions,therefore incorporation of appropriate substituents on the aromatic ringstructures, such as, by way of example only, halogens can reduce,minimize or eliminate this metabolic pathway.

Compounds described herein include isotopically-labeled compounds, whichare identical to those recited in the various formulas and structurespresented herein, but for the fact that one or more atoms are replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H,³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl, respectively. Certainisotopically-labeled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, areuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., ²H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

In additional or further embodiments, the compounds described herein aremetabolized upon administration to an organism in need to produce ametabolite that is then used to produce a desired effect, including adesired therapeutic effect.

Compounds described herein (for example, compounds of any of Formula(A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I),or Formula (VII)) are optionally in the form of, and/or used as,pharmaceutically acceptable salts. The type of pharmaceutical acceptablesalts, include, but are not limited to: (1) acid addition salts, formed)by reacting the free base form of the compound with a pharmaceuticallyacceptable: inorganic acid such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, metaphosphoric acid, andthe like; or with an organic acid such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoicacid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonicacid, 2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; (2) salts formed when anacidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion (e.g. lithium, sodium, potassium),an alkaline earth ion (e.g. magnesium, or calcium), or an aluminum ion;or coordinates with an organic base. Acceptable organic bases includeethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Acceptable inorganic bases includealuminum hydroxide, calcium hydroxide, potassium hydroxide, sodiumcarbonate, sodium hydroxide, and the like.

The corresponding counterions of the pharmaceutically acceptable saltsare optionally analyzed and identified using various methods including,but not limited to, ion exchange chromatography, ion chromatography,capillary electrophoresis, inductively coupled plasma, atomic absorptionspectroscopy, mass spectrometry, or any combination thereof.

The salts are recovered by using at least one of the followingtechniques: filtration, precipitation with a non-solvent followed byfiltration, evaporation of the solvent, or, in the case of aqueoussolutions, lyophilization.

It should be understood that a reference to a pharmaceuticallyacceptable salt includes the solvent addition forms or crystal formsthereof, particularly solvates or polymorphs. Solvates contain eitherstoichiometric or non-stoichiometric amounts of a solvent, and areoptionally formed during the process of crystallization withpharmaceutically acceptable solvents such as water, ethanol, and thelike. Hydrates are formed when the solvent is water, or alcoholates areformed when the solvent is alcohol. Solvates of compounds describedherein can be conveniently prepared or formed during the processesdescribed herein. In addition, the compounds provided herein can existin unsolvated as well as solvated forms. In general, the solvated formsare considered equivalent to the unsolvated forms for the purposes ofthe compounds and methods provided herein.

It should be understood that a reference to a salt includes the solventaddition forms or crystal forms thereof, particularly solvates orpolymorphs. Solvates contain either stoichiometric or non-stoichiometricamounts of a solvent, and are often formed during the process ofcrystallization with pharmaceutically acceptable solvents such as water,ethanol, and the like. Hydrates are formed when the solvent is water, oralcoholates are formed when the solvent is alcohol. Polymorphs includethe different crystal packing arrangements of the same elementalcomposition of a compound. Polymorphs usually have different X-raydiffraction patterns, infrared spectra, melting points, density,hardness, crystal shape, optical and electrical properties, stability,and solubility. Various factors such as the recrystallization solvent,rate of crystallization, and storage temperature may cause a singlecrystal form to dominate.

Compounds described herein are optionally in various forms, includingbut not limited to, amorphous forms, milled forms and nano-particulateforms. In addition, compounds described herein include crystallineforms, also known as polymorphs. Polymorphs include the differentcrystal packing arrangements of the same elemental composition of acompound. Polymorphs usually have different X-ray diffraction patterns,infrared spectra, melting points, density, hardness, crystal shape,optical and electrical properties, stability, and solubility. Variousfactors such as the recrystallization solvent, rate of crystallization,and storage temperature may cause a single crystal form to dominate.

The screening and characterization of the pharmaceutically acceptablesalts, polymorphs and/or solvates may be accomplished using a variety oftechniques including, but not limited to, thermal analysis, x-raydiffraction, spectroscopy, vapor sorption, and microscopy. Thermalanalysis methods address thermo chemical degradation or thermo physicalprocesses including, but not limited to, polymorphic transitions, andsuch methods are used to analyze the relationships between polymorphicforms, determine weight loss, to find the glass transition temperature,or for excipient compatibility studies. Such methods include, but arenot limited to, Differential scanning calorimetry (DSC), ModulatedDifferential Scanning calorimetry (MDCS), Thermogravimetric analysis(TGA), and Thermogravi-metric and Infrared analysis (TG/IR). X-raydiffraction methods include, but are not limited to, single crystal andpowder diffractometers and synchrotron sources. The variousspectroscopic techniques used include, but are not limited to, Raman,FTIR, UVIS, and NMR (liquid and solid state). The various microscopytechniques include, but are not limited to, polarized light microscopy,Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis(EDX), Environmental Scanning Electron Microscopy with EDX (in gas orwater vapor atmosphere), IR microscopy, and Raman microscopy.

Cysteine-Targeted Kinase Inhibitor Discovery Platform Kinases/InhibitorsSAR Approach

Protein kinases, which act on and modify the activity of specificproteins, are used to transmit signals and control complex processes incells. Up to 518 different kinases have been identified in humans. Manykinase inhibitor compounds non-selectively bind and/or inhibit thesekinases because the active sites of some of these kinases are similar instructure. Such cross-reactivity is not a desired feature of a kinaseinhibitor compound because of the potential for undesired side effectswhen such a compound is being administered to treat a disorder.

We have observed that small differences in the structure of kinaseinhibitor compounds have profound effects in the selectivity ofsimilarly-structured kinases (e.g., ACKs, including, Btk and the Btkkinase cysteine homologs).

As a result, we have developed assays, methods, and systems forconverting a non-selective inhibitor compound into a highly-selectiveinhibitor compound. In brief, the non-selective inhibitor compound isprovided with a Michael acceptor moiety and a linker moiety that linksthe Michael acceptor moiety to the remainder of the non-selectiveinhibitor compound. A series of linker and Michael acceptor moietiesprovides a small library/panel of test inhibitor compounds. Theinhibitor library/panel is contacted with a panel of structurallyrelated kinases (e.g., Btk and the Btk kinase cysteine homologs).Binding is determined by a variety of means, included fluorescencedetection (or via any other detectable label), mass spectrometry, or acombination of approaches. An Activity Probe is optionally used todetect binding of members of the inhibitor library/panel to the kinaselibrary/panel. The binding data is then optionally collected andanalyzed to provide a structure-activity relationship (SAR) between thestructure of the members of the inhibitor panel/library (e.g., Michaelacceptor and/or linker moieties) and the activity of binding to and/orinhibiting members of the kinase panel. Based on this information,further modifications are suggested if necessary. We have successfullyused this approach to improve the binding and selectivity of Btkinhibitor compounds (see Examples herein, including “Kinase InhibitorDiscovery Platform” example section).

In some embodiments, a similar approach is used for converting aselective inhibitor compound for a group of similarly-structured ACKs(including, Btk and the Btk kinase cysteine homologs) into a morehighly-selective inhibitor compound (e.g., more selective for aparticular ACK over structurally-similar ACKs), or for converting aselective inhibitor compound for a particular ACK (e.g., Btk) into aneven more selective inhibitor of that particular ACK. For example, inbrief, the selective inhibitor compound (which, for example, contains anactive-site binding moiety, a linker moiety and a Michael acceptormoiety) is modified. In one embodiment, a series of linker and Michaelacceptor moieties provides a small library/panel of test inhibitorcompounds. The inhibitor library/panel is contacted with a panel ofstructurally related kinases (e.g., Btk and the Btk kinase cysteinehomologs). Binding is determined by a variety of means, includedfluorescence detection (or via any other detectable label), massspectrometry, or a combination of approaches. An Activity Probe isoptionally used to detect binding of members of the inhibitorlibrary/panel to the kinase library/panel. The binding data is thenoptionally collected and analyzed to provide a structure-activityrelationship (SAR) between the structure of the members of the inhibitorpanel/library (e.g., Michael acceptor and/or linker moieties) and theactivity of binding to and/or inhibiting members of the kinase panel.Based on this information, further modifications are suggested ifnecessary. We have also successfully used this approach to improve thebinding and selectivity of Btk inhibitor compounds (see Examples herein,including “Kinase Inhibitor Discovery Platform” example section).

Thus, for our highly selective BTK inhibitor Compound 1, we engineeredan electrophilic center capable of irreversibly inactivating the targetenzyme, BTK. That is, to an active site binding moiety of a reversibleinhibitor was added a linker moiety and a Michael acceptor moiety thatachieved a high degree of potency and selectivity by (1) fitting thecore scaffold into the active site ATP binding pocket of kinase enzymes,and (2) forming a covalent bond with Cysteine-481 located in BTK. Thechemistry required for covalent bond formation involves an electrophilicmoiety that acts as a Michael acceptor, which bonds with a nucleophile(such as Cys-481) present in a precise location within the active site.

In another example, the linker and Michael acceptor moiety of Compound 1was modified to provide Compound 9 which has a different selectivitypattern. Table 1 is a table showing the degree of inhibition of a panelof kinases for two example compounds. IC₅₀s were determined using the invitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriatesubstrate and 1 uM ATP.) Compared to Compound 1, Compound 9 has similarpotency toward Btk, but significantly less potency toward JAK-3, ITK,and EGFR and significantly more potency toward the src-family kinaseslck, c-src, FGR, Fyn, Hck, and Lyn and Yes. Thus, subtle modificationsin the linker moiety and the Michael acceptor moiety are important forthe design of selective ACK inhibitors.

TABLE 1 Compound 1 Compound 9 Kinase IC50 (nM) IC50 (nM) BTK 0.5 1.0 ITK11.7 909.9 Bmx/ETK 0.8 1.1 TEC 77.8 108.0 EFGR 0.5 20.6 HER4 9.4 1536.0HER4 0.1 3.2 LCK 2.0 1.0 BLK 0.5 0.2 C-src 262.6 14.3 FGR 2.3 0.4 Fyn95.6 7.1 HCK 3.7 1.0 Lyn 16.2 1.2 YES 6.5 0.8 ABL 86.1 32.3 Brk 3.3 3.3CSK 2.2 2.4 FER 8,070.0 3,346.0 JAK3 10.4 8,278.0 SYK >10,000 >10,000

Table 2 of Example 1c in the “Kinase Discovery Platform and PulseDosing” section of the examples section provides further modificationsof the linker moiety and/or the Michael acceptor moiety and the impactof such changes of inhibitor selectivity.

Thus, in one aspect described herein are methods of identifying anirreversible inhibitor of a kinase selected from Btk, a Btk homolog, aBtk kinase cysteine homolog, an ACK, or HER4 (or indeed, any ACK)comprising:

(1) contacting a multiplicity of kinases selected from Btk, a Btkhomolog, a Btk kinase cysteine homolog, an ACK, or HER4 (or indeed anyACK) with a compound that comprises a Michael acceptor moiety;(2) contacting at least one non-kinase molecule having at least oneaccessible SH group with the compound that comprises a Michael acceptormoiety (this step allows for the selection of inhibitors that have lowselectivity for higher abundance biological molecules that have moietiesthat irreversibly react with the inhibitor; thus preventing theinhibitor from binding to the desire ACK when administered as a drug toan individual); and(3) determining the covalent binding of the compound that comprises aMichael acceptor with the multiplicity of kinases and the at least onenon-kinase molecule; and repeating steps (1), (2), and (3) for at leastone other compound that comprises a Michael acceptor moiety.

In a further aspect, the following steps are added: (4) comparing thecovalent binding of the compound that comprises a Michael acceptor withthe multiplicity of kinases and the at least one non-kinase molecule;and repeating steps (1), (2), (3) and (4) for at least one othercompound that comprises a Michael acceptor moiety.

In a further aspect the irreversible inhibitor compounds are alsocontacted with at least one non-ACK kinase in order to determine theselectivity of the irreversible inhibitor compound for the ACK relativeto the non-ACK.

By way of certain relevant examples of non-kinase molecules with atleast one accessible SH group are glutathione and/or hemoglobin. Becauseof the high abundance of these molecules in typical biological systems(e.g., in an individual), the desired irreversible inhibitor compoundshave low selectivity/reactivity with these non-kinase molecules.

In certain embodiments of the Kinase Inhibitor Discovery Platform, anActivity Probe (described in more detail herein) is used as a rapiddiagnostic method for determining whether a test inhibitor compound hasirreversibly inhibited an ACK. In one embodiment, the Activity Probe isitself an irreversible inhibitor of an ACK, an irreversible inhibitor ofHER4, and further, has a reporter moiety (e.g., a fluorescent moiety) aspart of its structure. When used in competition with a test irreversibleinhibitor, the absence of a ‘reporter’ signal on an ACK is oneindication that the test irreversible inhibitor has prevented theActivity Probe from binding to the ACK (and that the test irreversibleinhibitor has a higher binding affinity for the ACK than the ActivityProbe).

In certain embodiments, the Kinase Inhibitor Discovery Platform, steps(1) and (2) are conducted in vivo and step (3) is conducted in partusing an Activity Probe. Further, in certain embodiments, thedetermining step uses mass spectrometry, fluorescence, or a combinationthereof.

As described herein, in one embodiment, the inhibitor tested with theKinase Inhibitor Discovery Platform comprise an active site bindingmoiety, a Michael acceptor moiety, and a linker moiety that links theMichael acceptor moiety to the active site binding moiety. For example,in such a scheme, the following information is collected and analyzed:the structure-function activity relationship between the structure ofthe linker moiety and/or the Michael acceptor moiety of each compound,and the binding and/or selectivity of each compound to at least onekinase. Further, in certain embodiments, structure of the active sitebinding moiety of each compound is not varied, whereas the structure ofthe linker moiety and/or the Michael acceptor moiety is varied.

In one example, the inhibitors have the structure of Formula (VII):

wherein:

-   wherein

is a moiety that binds to the active site of a kinase, including atyrosine kinase, further including a Btk kinase cysteine homolog;

-   Y is an optionally substituted group selected from among alkylene,    heteroalkylene, arylene, heteroarylene, heterocycloalkylene,    cycloalkylene, alkylenearylene, alkyleneheteroarylene,    alkylenecycloalkylene, and alkyleneheterocycloalkylene;-   Z is C(═O), OC(═O), NHC(═O), NCH₃C(═O), C(═S), S(═O)_(x),    OS(═O)_(x), NHS(═O)_(x), where x is 1 or 2;-   R₇ and R₈ are independently selected from among H, unsubstituted    C₁-C₄ alkyl, substituted C₁-C₄alkyl, unsubstituted C₁-C₄heteroalkyl,    substituted C₁-C₄heteroalkyl, unsubstituted C₃-C₆cycloalkyl,    substituted C₃-C₆cycloalkyl, unsubstituted C₂-C₆heterocycloalkyl,    and substituted C₂-C₆heterocycloalkyl; or-   R₇ and R₈ taken together form a bond; and-   R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted or    unsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl,    C₁-C₈alkylaminoalkyl, C₁-C₈hydroxyalkylaminoalkyl,    C₁-C₈alkoxyalkylaminoalkyl, substituted or unsubstituted    C₃-C₆cycloalkyl, substituted or unsubstituted aryl, substituted or    unsubstituted C₂-C₈heterocycloalkyl, substituted or unsubstituted    heteroaryl, C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl),    C₁-C₄alkyl(C₃-C₈cycloalkyl), or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).

In such a scheme, the following information is collected and analyzed:the structure-function activity relationship between the structure ofY—Z and/or

of each compound, and the binding and/or selectivity of each compound toat least one kinase. Further, the structure of

of each compound is not varied, whereas the structure of the linkermoiety (Y—Z) and/or the Michael acceptor moiety

is varied.

In certain embodiments of the Kinase Inhibitor Discovery Platform, theresulting inhibitor is selective for one kinase selected from Btk, a Btkhomolog, an ACK, HER4, and a Btk kinase cysteine homolog over at leastone other kinase selected from Btk, a Btk homolog, an ACK, HER4, and aBtk kinase cysteine homolog. In some embodiments, this selectivity is atleast 5×, at least 10×, at least 20×, at least 50×, or at least 100×. Infurther embodiments, the resulting inhibitor is selective for at leastone kinase selected from Btk, a Btk homolog, an ACK, HER4, and a Btkkinase cysteine homolog over at least one other non-kinase moleculehaving an accessible SH group. In some embodiments, this selectivity isat least 5×, at least 10×, at least 20×, at least 50×, or at least 100×.

In further embodiments, the resulting inhibitor is used in thetherapeutic methods described herein, or in the pharmaceuticalcompositions described herein.

Activity Probe Compounds

Because of the Kinase Inhibitor Discovery Platform described hereinoptionally utilizes an Activity Probe, the following section describesthe design, structure and use of non-limiting examples of ActivityProbes.

The Activity Probe compounds described herein are composed of a moietycomprising an inhibitor of Btk, a Btk homolog, a Btk kinase cysteinehomolog, an ACK, or HER4 (hereinafter, a “Kinase Inhibitor”), a linkermoiety, and a reporter moiety. In one embodiment, the Kinase Inhibitoris an irreversible inhibitor. In another embodiment, the irreversibleKinase Inhibitor binds to a non-catalytic residue in the ATP bindingpocket of Btk, a Btk homolog, a Btk kinase cysteine homolog, an ACK, orHER4 (hereinafter a “Kinase”); in further embodiments, the non-catalyticresidue is a cysteine residue. In some embodiments, the Activity Probeforms a covalent bond with at least one non-catalytic residue of aKinase. In other embodiments, the Activity Probe forms a non-covalentbond with at least one non-catalytic residue of a Kinase. In a furtherembodiment, the Activity Probe forms hydrogen bonding within the ATPbinding pocket of a Kinase. In yet a further embodiment, the ActivityProbe has Van der Waals attractions with the Kinase.

In some other embodiments, the Activity Probes described herein areactivity dependent such that the probe binds only an active Kinase. Infurther embodiments, the Activity Probe binds a Kinase that has beenswitched on by phosphorylation by upstream kinases. In yet a furtherembodiment, the Activity Probes described herein are activityindependent such that the probe binds Kinases that have not beenswitched on by phosphorylation by upstream kinases. In some embodiments,the Activity Probe labels a phosphorylated conformation of a Kinase. Inother embodiments, the Activity Probe labels a Kinase in anon-phosphorylated conformation.

In some embodiments, the Activity Probe is permeable to cells.

In further embodiments, the linker moiety is selected from a bond, asubstituted alkyl moiety, a substituted heterocycle moiety, asubstituted amide moiety, a ketone moiety, a substituted carbamatemoiety, an ester moiety, or any combination thereof. In furtherembodiments, the reporter moiety is a moiety that is detected usingstandard or modified laboratory equipment.

In one aspect is a Activity Probe of Formula (I) comprising:

wherein:

-   -   A is a Kinase Inhibitor moiety;    -   X and Y are independently selected from the group consisting of:        a bond, —O(C═O)—, —NR^(a)(C═O)—, —NR^(a)—,

—O—, —S—, —S—S—, —O—NR^(a)—, —O(C═O)O—, —O(C═O)NR^(a),—NR^(a)(C═O)NR^(a)—, —N═CR^(a)—, —S(C═O)—, —S(O)—, and —S(O)₂—;

-   -   wherein

forms a N-containing heterocycle;

-   -   B is a linker moiety;    -   C is a reporter moiety; and    -   R^(a) is hydrogen or alkyl.

In one embodiment, the moiety comprising an irreversible KinaseInhibitor is derived from an irreversible inhibitor of a Kinase. In someembodiments, such irreversible Kinase Inhibitors should possess at leastone of the following characteristics: potency, selectively and cellpermeability. In further embodiments, such irreversible KinaseInhibitors possess at least two of the aforementioned characteristics,and in further embodiments, at least all of the aforementionedcharacteristics.

In another embodiment, the Kinase Inhibitor moiety is derived from a Btkinhibitor having the structure of Formula (II):

wherein:

-   L_(a) is CH₂, O, NH or S;-   Ar is a substituted or unsubstituted aryl, or a substituted or    unsubstituted heteroaryl; and-   Y is an optionally substituted group selected from among alkylene,    heteroalkylene, arylene, heteroarylene, heterocycloalkylene,    cycloalkylene, alkylenearylene, alkyleneheteroarylene,    alkylenecycloalkylene, and alkyleneheterocycloalkylene.

In some embodiments, L_(a) is CH₂O, or NH. In other embodiments, L_(a)is O or NH. In yet other embodiments, L_(a) is O.

In other embodiments, Ar is a substituted or unsubstituted aryl. In yetother embodiments, Ar is a 6-membered aryl. In some other embodiments,Ar is phenyl.

In some embodiments, Y is an optionally substituted group selected fromamong alkylene, heteroalkylene, arylene, heteroarylene,heterocycloalkylene, cycloalkylene, alkylenearylene,alkyleneheteroarylene, alkylenecycloalkylene, andalkyleneheterocycloalkylene. In other embodiments, Y is an optionallysubstituted group selected from among C₁-C₆alkylene,C₁-C₆heteroalkylene, 4-, 5-, 6-, or 7-membered cycloalkylene, and 4-,5-, 6-, or 7-membered heterocycloalkylene. In yet other embodiments, Yis an optionally substituted group selected from among C₁-C₆alkylene,C₁-C₆heteroalkylene 5- or 6-membered cycloalkylene, and 5- or 6-memberedheterocycloalkylene containing 1 or 2 N atoms. In some otherembodiments, Y is a 5- or 6-membered cycloalkylene, or a 5- or6-membered heterocycloalkylene containing 1 or 2 N atoms. In someembodiments, Y is a 4-, 5-, 6-, or 7-membered cycloalkylene ring; or Yis a 4-, 5-, 6-, or 7-membered heterocycloalkylene ring.

In some embodiments, the Kinase Inhibitor moiety is derived from acompound selected from among:1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one;(E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)but-2-en-1-one;1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)sulfonylethene;1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-yn-1-one;1-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one;N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)acrylamide;1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one;1-((S)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one;1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one;1-((S)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one;and(E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-(dimethylamino)but-2-en-1-one;(E)-4-(N-(2-hydroxyethyl)-N-methylamino)-1-(3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)but-2-en-1-one(Compound 3);(E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-(1H-imidazol-4-yl)prop-2-en-1-one(Compound 4);(E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-morpholinobut-2-en-1-one(Compound 5);(E)-1-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-(dimethylamino)but-2-en-1-one(Compound 7);(E)-N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)-4-(dimethylamino)but-2-enamide(Compound 8);N-((1r,4r)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)acrylamide(Compound 10);(E)-1-((R)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrolidin-1-yl)-4-(dimethylamino)but-2-en-1-one(Compound 11);(E)-1-((S)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrolidin-1-yl)-4-(dimethylamino)but-2-en-1-one(Compound 12);1-((R)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)prop-2-en-1-one(Compound 13);1-((S)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)prop-2-en-1-one(Compound 14);1((R)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)but-2-yn-1-one(Compound 15);1-((S)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)but-2-yn-1-one(Compound 16);1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)but-2-yn-1-one(Compound 17);(E)-N-((1,r,4r)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl-4-(dimethylamino)but-2-enamide(Compound 18);N-(2-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-methylacrylamide(Compound 19);(E)-1-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-morpholinobut-2-en-1-one(Compound 20); (E)-1-((Samino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)-4-morpholinobut-2-en-1-one(Compound 21);N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)but-2-ynamide(Compound 22);N-(2-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)acrylamide(Compound 23);(E)-1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-morpholinobut-2-en-1-one(Compound 24); (E)-N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)-4-morpholinobut-2-enamide(Compound 25).

In another embodiment, the linker moiety is selected from a bond, apolymer, a water soluble polymer, optionally substituted alkyl,optionally substituted heteroalkyl, optionally substitutedheterocycloalkyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkylalkyl, optionally substitutedheterocycloalkylalkenyl, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substitutedheterocycloalkylalkenylalkyl. In some embodiments, the linker moiety isan optionally substituted heterocycle. In other embodiments, theheterocycle is selected from aziridine, oxirane, episulfide, azetidine,oxetane, pyrroline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine,pyrazole, pyrrole, imidazole, triazole, tetrazole, oxazole, isoxazole,oxirene, thiazole, isothiazole, dithiolane, furan, thiophene,piperidine, tetrahydropyran, thiane, pyridine, pyran, thiapyrane,pyridazine, pyrimidine, pyrazine, piperazine, oxazine, thiazine,dithiane, and dioxane. In some embodiments, the heterocycle ispiperazine. In further embodiments, the linker moiety is optionallysubstituted with halogen, CN, OH, NO₂, alkyl, S(O), and S(O)₂. In otherembodiments, the water soluble polymer is a PEG group.

In other embodiments, the linker moiety provides sufficient spatialseparation between the reporter moiety and the Kinase Inhibitor moiety.In further embodiments, the linker moiety is stable. In yet a furtherembodiment, the linker moiety does not substantially affect the responseof the reporter moiety. In other embodiments the linker moiety provideschemical stability to the Activity Probe. In further embodiments, thelinker moiety provides sufficient solubility to the Activity Probe.

In some embodiments, linkages such as water soluble polymers are coupledat one end to a Kinase Inhibitor moiety and to a reporter moiety at theother end. In other embodiments, the water soluble polymers are coupledvia a functional group or substituent of the Kinase Inhibitor moiety. Infurther embodiments, the water soluble polymers are coupled via afunctional group or substituent of the reporter moiety. In otherembodiments, covalent attachment of hydrophilic polymers to a KinaseInhibitor moiety and a reporter moiety represents one approach toincreasing water solubility (such as in a physiological environment),bioavailability, increasing serum half-life, increasing pharmacodynamicparameters, or extending the circulation time of the Activity Probe,including proteins, peptides, and particularly hydrophobic molecules. Infurther embodiments, additional important features of such hydrophilicpolymers include biocompatibility and lack of toxicity. In otherembodiments, for therapeutic use of the end-product preparation, thepolymer is pharmaceutically acceptable.

In some embodiments, examples of hydrophilic polymers include, but arenot limited to: polyalkyl ethers and alkoxy-capped analogs thereof(e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol, andmethoxy or ethoxy-capped analogs thereof, polyoxyethylene glycol, thelatter is also known as polyethylene glycol or PEG);polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyloxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkylacrylamides, and polyhydroxyalkyl acrylamides (e.g.,polyhydroxypropylmethacrylamide and derivatives thereof);polyhydroxyalkyl acrylates; polysialic acids and analogs thereof;hydrophilic peptide sequences; polysaccharides and their derivatives,including dextran and dextran derivatives, e.g., carboxymethyldextran,dextran sulfates, aminodextran; cellulose and its derivatives, e.g.,carboxymethyl cellulose, hydroxyalkyl celluloses; chitin and itsderivatives, e.g., chitosan, succinyl chitosan, carboxymethylchitin,carboxymethylchitosan; hyaluronic acid and its derivatives; starches;alginates; chondroitin sulfate; albumin; pullulan and carboxymethylpullulan; polyaminoacids and derivatives thereof, e.g., polyglutamicacids, polylysines, polyaspartic acids, polyaspartamides; maleicanhydride copolymers such as: styrene maleic anhydride copolymer,divinylethyl ether maleic anhydride copolymer; polyvinyl alcohols;copolymers thereof; terpolymers thereof; mixtures thereof; andderivatives of the foregoing. In other embodiments, the water solublepolymer is any structural form including but not limited to linear,forked or branched. In some embodiments, polymer backbones that arewater-soluble, with from 2 to about 300 termini, are particularlyuseful. In further embodiments, multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which is the same ordifferent. In some embodiments, the water polymer comprises apoly(ethylene glycol) moiety. In further embodiments, the molecularweight of the polymer is of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. In yet furtherembodiments, the molecular weight of the polymer is between about 100 Daand about 100,000 Da, including but not limited to, about 100,000 Da,about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da,about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da,about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da,about 35,000 Da, 30,000 Da, about 25,000 Da, about 20,000 Da, about15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da,about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700Da, about 600 Da, about 500 Da, about 400 Da, about 300 Da, about 200Da, and about 100 Da. In some embodiments, the molecular weight of thepolymer is between about 100 Da and 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and 40,000 Da. In some embodiments, the poly(ethylene glycol)molecule is a branched polymer. In further embodiments, the molecularweight of the branched chain PEG is between about 1,000 Da and about100,000 Da, including but not limited to, about 100,000 Da, about 95,000Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da,about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da,about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da,about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da,about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000Da, and about 1,000 Da. In some embodiments, the molecular weight of thebranched chain PEG is between about 1,000 Da and about 50,000 Da. Insome embodiments, the molecular weight of the branched chain PEG isbetween about 1,000 Da and about 40,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 5,000 Da andabout 40,000 Da. In some embodiments, the molecular weight of thebranched chain PEG is between about 5,000 Da and about 20,000 Da. Theforegoing list for substantially water soluble backbones is by no meansexhaustive and is merely illustrative, and in some embodiments, thepolymeric materials having the qualities described above suitable foruse in methods and compositions described herein.

In further embodiments, the number of water soluble polymers linked to aKinase Inhibitor moiety and a reporter moiety described herein isadjusted to provide an altered (including but not limited to, increasedor decreased) pharmacologic, pharmacokinetic or pharmacodynamiccharacteristic such as in vivo half-life. In some embodiments, thehalf-life of the Activity Probe is increased at least about 10, about20, about 30, about 40, about 50, about 60, about 70, about 80, about 90percent, about two fold, about five-fold, about 10-fold, about 50-fold,or at least about 100-fold over a Activity Probe without a water solublelinker.

In another embodiment, X is selected from the group consisting of: abond, —O(C═O)—, —NR^(a)(C═O)—, —NR^(a)—,

—O—, —S—, —S—S—, —O(C═O)O—, —O(C═O)NR^(a), —NR^(a)(c=O)NR^(a)—,—N═CR^(a)—, —S(C═O)—, —S(O)—, and —S(O)₂—; wherein

forms a N-containing heterocycle. In one embodiment, X is NR^(a)(C═O).In another embodiment, X is a bond. In another embodiment, X is—O(C═O)—. In a further embodiment, Y is selected from the groupconsisting of: a bond, —O(C═O)—, —NR^(a)(C═O)—, —NR^(a)—,

—O—, —S—, —S—S—, —O—NR^(a)—, —O(C═O)O—, —O(C═O)NR^(a),—NR^(a)(C═O)NR^(a)—, —N═CR^(a)—, —S(C═O)—, —S(O)—, and —S(O)₂—; wherein

forms a N-containing heterocycle. In yet a further embodiment, Y is abond. In one embodiment, Y is —NR^(a)(C═O)—. In yet another embodiment,R^(a) is hydrogen. In yet a further embodiment, R^(a) is alkyl.

In a further embodiment, the reporter moiety is selected from the groupconsisting of a label, a dye, a photocrosslinker, a cytotoxic compound,a drug, an affinity label, a photoaffinity label, a reactive compound,an antibody or antibody fragment, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a ligand, a photoisomerizable moiety,biotin, a biotin analog, a moiety incorporating a heavy atom, achemically cleavable group, a photocleavable group, a redox-activeagent, an isotopically labeled moiety, a biophysical probe, aphosphorescent group, a chemiluminescent group, an electron dense group,a magnetic group, an intercalating group, a chromophore, an energytransfer agent, a biologically active agent, a detectable label, or acombination thereof.

In another embodiment, the reporter moiety is a fluorophore. In afurther embodiment, the fluorophore is selected from the groupconsisting of: BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550,BODIPY TMR, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY TR, Fluorescein, 5(6)-Carboxyfluorescein,2,7-Dichlorofluorescein,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide, HPTS,Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTO 465, ATTO 488, ATTO 495, YOYO-1, 5-FAM, BCECF, BCECF,dichlorofluorescein, rhodamine 110, rhodamine 123, Rhodamine Green,YO-PRO-1, SYTOX Green, Sodium Green, SYBR Green I, Alexa Fluor 500,FITC, Fluo-3, Fluo-4, fluoro-emerald, YoYo-1 ssDNA, YoYo-1 dsDNA,YoYo-1, SYTO RNASelect, Diversa Green-FP, Dragon Green, EvaGreen, SurfGreen EX, Spectrum Green, Oregon Green 488, NeuroTrace 500525, NBD-X,MitoTracker Green FM, LysoTracker Green DND-26, CBQCA, PA-GFP(post-activation), WEGFP (post-activation), FlASH-CCXXCC, Azami Greenmonomeric, Azami Green, EGFP (Campbell Tsien 2003), EGFP (Patterson2001), Fluorescein, Kaede Green,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bexl, Doxorubicin, LumioGreen, and SuperGlo GFP.

In a further embodiment, the fluorophore is selected from the groupconsisting of: BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550,BODIPY TMR, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, and BODIPY TR. In yet a further embodiment, the fluorophore isBODIPY FL. In certain embodiments, the fluorophore is not BODIPY 530. Insome embodiments, the fluorophore has an excitation maxima of betweenabout 500 and about 600 nm. In some other embodiments, the fluorophorehas an excitation maxima of between about 500 and about 550 nm. Inanother embodiments, the fluorophore has an excitation maxima of betweenabout 550 and about 600 nm. In yet a further embodiment, the fluorophorehas an excitation maxima of between about 525 and about 575 nm. In otherembodiments, the fluorophore has an emission maxima of between about 510and about 670 nm. In another embodiment, the fluorophore has an emissionmaxima of between about 510 and about 600 nm. In a further embodiment,the fluorophore has an emission maxima of between about 600 and about670 nm. In another embodiment, the fluorophore has an emission maxima ofbetween about 575 and about 625 nm.

By way of example only and in some embodiments, the observed potency,selectivity, and cell permeability of compounds such as Compound 2 areappropriate to incorporate these molecules into a Kinase-targeted,activity based probe that allows direct visualization of Kinase activityin intact cells. In vitro profiling against a panel of greater than 100kinases showed Compound 2 to be a highly potent and selective inhibitorof Tec family kinases, including, Btk, as well as Src family kinases.Without limiting the scope of the compositions and methods describedherein, it is postulated that the structural basis for the selectivityis covalent modification of a non-catalytic cysteine residue (Cys 481 inBtk) that is conserved in the ATP binding pocket of the Tec family andseveral other kinases.

However, in other embodiments, any irreversible Kinase Inhibitor thatbinds to the non-catalytic cysteine residue in the ATP binding pocket ofa Kinase is used in the compounds and methods described herein.

General Synthesis and Characterization of an Illustrative Activity Probe

Without limiting the scope of the compositions described herein, anillustrative probe was synthesized by attaching a bodipy FL fluorophoreto an irreversible inhibitor via a piperazine linker. The piperazinelinker served to maintain probe solubility and provided spatialseparation between the fluorophore and the pyrazolopyrimidine core.

In some embodiments, the linkage formed is a stable linkage. In otherembodiments, in the case where the conjugate comprises two components,the linker moiety forms a linkage, in some embodiments, a stablelinkage, between the Kinase Inhibitor moiety and the reporter moiety. Insome embodiments, the linker moiety is stable and provides the means tocontrol and determine the distance between the Kinase Inhibitor moietyand the report moiety. Further, in some embodiments, the linker moietyis selected such that the probe's solubility is maintained. In someembodiments, the linker moiety is a piperazinyl moiety. In furtherembodiments, a piperazinyl-based linkage is formed by using a piperazinecontaining compound. In other embodiments, the number and order of unitsthat comprise the linker moiety is selected such that the length betweenthe first component and the second component, as well as the hydrophobicand hydrophilic characteristics of the linker is controlled.

In the present context, spatial separation means a thermochemically andphotochemically non-active distance-making group and in some embodimentsis used to join two or more different moieties of the types definedabove. In other embodiments, spacers are selected on the basis of avariety of characteristics including their hydrophobicity,hydrophilicity, molecular flexibility and length. The spacer, thus, insome embodiments, comprises a chain of carbon atoms optionallyinterrupted or terminated with one or more heteroatoms, such as oxygenatoms, nitrogen atoms, and/or sulphur atoms. Thus, in some embodiments,the spacer comprises one or more amide, ester, amino, ether, and/orthioether functionalities, and optionally aromatic ormono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethyleneglycol, oligo/polyamides such as poly-.α-alanine, polyglycine,polylysine, and peptides in general, oligosaccharides,oligo/polyphosphates. Moreover, in other embodiments, the spacerconsists of combined units thereof. In further embodiments, the lengthof the spacer varies, taking into consideration the desired or necessarypositioning and spatial orientation of the active/functional part of theActivity Probe.

Without limiting the scope of the compositions described herein, in someembodiments the reporter moiety is Bodipy. In the present context, theterm reporter moiety means a group which is detectable either by itselfor as a part of a detection series.

In some embodiments, the labeled Activity Probes described herein arepurified by one or more procedures including, but are not limited to,affinity chromatography; anion- or cation-exchange chromatography(using, including but not limited to, DEAE SEPHAROSE); chromatography onsilica; reverse phase HPLC; gel filtration (using, including but notlimited to, SEPHADEX G-75); hydrophobic interaction chromatography;size-exclusion chromatography, metal-chelate chromatography;ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), or extraction. In otherembodiments, apparent molecular weight is estimated by GPC by comparisonto globular protein standards (PROTEIN PURIFICATION METHODS, A PRACTICALAPPROACH (Harris & Angal, Eds.) IRL Press 1989, 293-306).

In one aspect, the in vitro inhibitory potency of a probe against apanel of selected Kinases as a rapid means of confirming accessibilityof the reactive moiety to the Kinase active site is tested. By way ofexample only, although less potent than the parent Compound 2, theillustrative probe of Compound 3 retains potency against Btk (IC₅₀˜90nM). Thus, the piperazine linker and bodipy fluorophore do not seriouslycompromise accessibility of the illustrative probe to the enzyme activesite.

The Activity Probes described herein label kinases at the non-catalyticCys 481 (or a homologous cysteine) and that in some embodiments, probelabeling does not require the catalytic machinery per se. As such itdiffers from canonical activity-based probes that target the enzymecatalytic machinery directly. In some embodiments, the Kinase undergoesa phosphorylation dependent conformational change that is tightlycoupled to ATP binding and kinase activation. In some embodiments,effective labeling by a probe requires the Kinase to be in its activeconformation in order to directly detect Kinase activity in cells. Inother embodiments, effective labeling by an Activity Probe does notrequire the Kinase to be in its active conformation in order to directlydetect Kinase activity in cells.

Therapeutic Uses of Irreversible Inhibitor Compounds

Described herein are methods, compositions, uses and medicaments for thetreatment of disorders characterized by the presence of a solid tumorcomprising administering to an individual in need an irreversibleinhibitor of an ACK. In some embodiments, the disorder is a sarcoma,lymphoma, and/or carcinoma. In some embodiments, the disorder is mammaryductal carcinoma, lobular carcinoma, an adenocarcinoma (e.g. pancreaticcancer and colon cancer), small cell lung carcinoma, non-small cell lungcarcinoma, and melanomas. In some embodiments, the disorder is mammaryductal carcinoma, lobular carcinoma, or a combination thereof. In someembodiments, the disorder is pancreatic cancer.

In some embodiments, the ACK is Btk or a Btk homolog. In yet furtherembodiments, the ACK is tyrosine kinases that share homology with Btk byhaving a cysteine residue (including a Cys 481 residue) that forms acovalent bond with the irreversible inhibitor. See, e.g., proteinkinases in FIG. 7. In some embodiments, the ACK is HER4.

The methods described herein (which includes uses of a pharmaceuticalcomposition to treat a disorder, or uses of a compound to form amedicament for treating a disorder) include administering to anindividual in need thereof a composition containing a therapeuticallyeffective amount of one or more irreversible Btk inhibitor compoundsdescribed herein. In some embodiments, the individual has been diagnosedwith or is predisposed to develop a sarcoma, lymphoma, and/or carcinoma.In some embodiments, the individual has been diagnosed with or ispredisposed to develop mammary ductal carcinoma, lobular carcinoma, anadenocarcinoma (e.g. pancreatic cancer and colon cancer), small celllung carcinoma, non-small cell lung carcinoma, and melanomas. In someembodiments, the individual has been diagnosed with or is predisposed todevelop mammary ductal carcinoma, lobular carcinoma, or a combinationthereof. In some embodiments, the individual has been diagnosed with oris predisposed to develop pancreatic cancer.

Without being bound by theory, the diverse roles played by Btk signalingin various hematopoietic cell functions show that small molecule Btkinhibitors are useful for reducing the risk of or treating a disordercharacterized by the presence or development of one or more solidtumors.

In some embodiments, are methods for treating a disorder characterizedby the presence of a solid tumor (e.g. lymphomas, carcinomas, and/orsarcomas) comprising administering to an individual in need apharmaceutical formulation of any irreversible inhibitor of Btk (or aBtk homolog) of Formula (A1-A6), Formula (B1-B6), Formula (C1-C6),Formula (D1-D6), Formula (I), or Formula (VII). In some embodiments, thesolid neoplasm is mammary ductal carcinoma, lobular carcinoma, anadenocarcinoma (e.g. pancreatic cancer and colon cancer), small celllung carcinoma, non-small cell lung carcinoma, and melanomas.

In still further embodiments are methods for treating a disordercharacterized by the presence of a solid tumor comprising administeringto an individual in need thereof a composition containing atherapeutically effective amount of a compound that forms a covalentbond with a cysteine sidechain of a Bruton's tyrosine kinase or aBruton's tyrosine kinase homolog. In some embodiments, the individualhas been diagnosed with or is predisposed to develop a sarcoma,lymphoma, and/or carcinoma. In some embodiments, the individual has beendiagnosed with or is predisposed to develop mammary ductal carcinoma,lobular carcinoma, an adenocarcinoma (e.g. pancreatic cancer and coloncancer), small cell lung carcinoma, non-small cell lung carcinoma, andmelanomas. In some embodiments, the individual has been diagnosed withor is predisposed to develop mammary ductal carcinoma, lobularcarcinoma, or a combination thereof. In some embodiments, the individualhas been diagnosed with or is predisposed to develop pancreatic cancer.

Further, in some embodiments, the irreversible Btk inhibitor compoundsdescribed herein are used to inhibit a small subset of other tyrosinekinases that share homology with Btk by having a cysteine residue(including a Cys 481 residue) that is able to form a covalent bond withthe irreversible inhibitor. See, e.g., protein kinases in FIG. 7. Thus,a subset of tyrosine kinases other than Btk are also expected to beuseful as therapeutic targets in a number of health conditions,including lymphomas, carcinomas, and/or sarcomas.

Symptoms, diagnostic tests, and prognostic tests for each of theabove-mentioned conditions include, e.g., Harrison's Principles ofInternal Medicine©,” 16th ed., 2004, The McGraw-Hill Companies, Inc. Deyet al. (2006), Cytojournal 3(24), and the “Revised European AmericanLymphoma” (REAL) classification system (see, e.g., the websitemaintained by the National Cancer Institute).

A number of animal models are useful for establishing a range oftherapeutically effective doses of irreversible inhibitors, includingirreversible Btk inhibitor compounds for treating any of the foregoingdiseases. For example, refer to Examples 1-4 of the “Therapeutic Uses”section of the Examples included herein. As an example, dosing ofirreversible inhibitors for the treatment of cancer can be examined in,e.g., a human-to-mouse xenograft model in which human B-cell lymphomacells (e.g. Ramos cells) are implanted into immunodefficient mice (e.g.,“nude” mice) as described in, e.g., Pagel et al. (2005), Clin Cancer Res11(13):4857-4866. Animal models for treatment of thromboembolicdisorders are also known.

In one embodiment, the therapeutic efficacy of the compound for one ofthe foregoing diseases is optimized during a course of treatment. Forexample, an individual being treated optionally undergoes a diagnosticevaluation to correlate the relief of disease symptoms or pathologies toinhibition of in vivo Btk activity achieved by administering a givendose of an irreversible Btk inhibitor. Cellular assays are used todetermine in vivo activity of Btk in the presence or absence of anirreversible Btk inhibitor. For example, since activated Btk isphosphorylated at tyrosine 223 (Y223) and tyrosine 551 (Y551),phospho-specific immunocytochemical staining of P-Y223 orP-Y551-positive cells are used to detect or quantify activation of Bktin a population of cells (e.g., by FACS analysis of stained vs unstainedcells). See, e.g., Nisitani et al. (1999), Proc. Natl. Acad. Sci, USA96:2221-2226. Thus, the amount of the Btk inhibitor compound that isadministered to an individual is optionally increased or decreased asneeded so as to maintain a level of Btk inhibition optimal for treatingthe subject's disease state.

In one embodiment are methods for identifying biomarkers suitable fordetermining patient response to an irreversible ACK inhibitor(including, e.g., a compound of Formula (I)) comprising administering toa test subject a composition containing an amount of the irreversibleACK inhibitor (including, e.g., a compound of Formula (I)) sufficient toinhibit B cell receptor signaling and correlating B cell receptorsignaling with apoptosis. In another or further embodiment are methodsfor selecting an individual for treatment for lymphoma with anirreversible ACK inhibitor (including, e.g., a compound of Formula (I))comprising measuring pErk or Erk transcriptional target levels in anindividual sample, and correlating a high level of transcriptionaltargets with a positive response to the treatment. In another or furtherembodiments are methods for measuring an individual's response totreatment comprising administering to the patient an irreversible ACKinhibitor (including, e.g., a compound of Formula (I)), measuring pErkor Erk transcriptional target levels in an individual sample, andcorrelating a reduced level of transcriptional targets with a positiveresponse to the administration of the irreversible ACK inhibitor(including, e.g., a compound of Formula (I)).

Combination Treatments

In some embodiments, the irreversible Btk inhibitor compositionsdescribed herein are used in combination with other well knowntherapeutic reagents that are selected for their therapeutic value forthe condition to be treated. In general, the compositions describedherein and, in embodiments where combinational therapy is employed,other agents do not have to be administered in the same pharmaceuticalcomposition, and are optionally, because of different physical andchemical characteristics, have to be administered by different routes.The initial administration is made, for example, according toestablished protocols, and then, based upon the observed effects, thedosage, modes of administration and times of administration aremodified.

In certain instances, it is appropriate to administer at least oneirreversible Btk inhibitor compound described herein in combination withanother therapeutic agent. By way of example only, if one of the sideeffects experienced by an individual upon receiving one of theirreversible Btk inhibitor compounds described herein is nausea, then itis appropriate to administer an anti-nausea agent in combination withthe initial therapeutic agent. Or, by way of example only, thetherapeutic effectiveness of one of the compounds described herein isenhanced by administration of an adjuvant (i.e., by itself the adjuvanthas minimal therapeutic benefit, but in combination with anothertherapeutic agent, the overall therapeutic benefit to the patient isenhanced). Or, by way of example only, the benefit experienced by anindividual is increased by administering one of the compounds describedherein with another therapeutic agent (which also includes a therapeuticregimen) that also has therapeutic benefit. In any case, regardless ofthe disease, disorder being treated, the overall benefit experienced bythe patient is in some embodiments simply additive of the twotherapeutic agents or in other embodiments, the patient experiences asynergistic benefit.

The particular choice of compounds used will depend upon the diagnosisof the attending physicians and their judgment of the condition of thepatient and the appropriate treatment protocol. The compounds areoptionally administered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,depending upon the nature of the disorder, the condition of the patient,and the actual choice of compounds used. The determination of the orderof administration, and the number of repetitions of administration ofeach therapeutic agent during a treatment protocol, is based on anevaluation of the disease being treated and the condition of thepatient.

In some embodiments, therapeutically-effective dosages vary when thedrugs are used in treatment combinations. Methods for experimentallydetermining therapeutically-effective dosages of drugs and other agentsfor use in combination treatment regimens are described in theliterature. For example, the use of metronomic dosing, i.e., providingmore frequent, lower doses in order to minimize toxic side effects, hasbeen described extensively in the literature Combination treatmentfurther includes periodic treatments that start and stop at varioustimes to assist with the clinical management of the patient.

For combination therapies described herein, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the disorder beingtreated and so forth. In addition, when co-administered with one or morebiologically active agents, the compound provided herein is administeredeither simultaneously with the biologically active agent(s), orsequentially. If administered sequentially, the attending physician willdecide on the appropriate sequence of administering protein incombination with the biologically active agent(s).

In any case, the multiple therapeutic agents (one of which is a compoundof Formula (A1-A6), (B1-B6), (C1-C6), or (D1-D6) described herein) areoptionally administered in any order or even simultaneously. Ifsimultaneously, the multiple therapeutic agents are optionally providedin a single, unified form, or in multiple forms (by way of example only,either as a single pill or as two separate pills). In some embodiments,one of the therapeutic agents is given in multiple doses, or both aregiven as multiple doses. If not simultaneous, the timing between themultiple doses is from about more than zero weeks to less than aboutfour weeks. In addition, the combination methods, compositions andformulations are not to be limited to the use of only two agents; theuse of multiple therapeutic combinations are also envisioned.

It is understood that the dosage regimen to treat, prevent, orameliorate the condition(s) for which relief is sought, can be modifiedin accordance with a variety of factors. These factors include thedisorder from which the subject suffers, as well as the age, weight,sex, diet, and medical condition of the subject. Thus, the dosageregimen actually employed can vary widely and therefore can deviate fromthe dosage regimens set forth herein.

In some embodiments, the pharmaceutical agents which make up thecombination therapy disclosed herein are administered in a combineddosage form, or in separate dosage forms intended for substantiallysimultaneous administration. In some embodiments, the pharmaceuticalagents that make up the combination therapy are administeredsequentially, with either therapeutic compound being administered by aregimen calling for two-step administration. In some embodiments, thetwo-step administration regimen calls for sequential administration ofthe active agents or spaced-apart administration of the separate activeagents. The time period between the multiple administration steps rangesfrom a few minutes to several hours, depending upon the properties ofeach pharmaceutical agent, such as potency, solubility, bioavailability,plasma half-life and kinetic profile of the pharmaceutical agent. Insome embodiments, circadian variation of the target moleculeconcentration determines the optimal dose interval.

In addition, the compounds described herein also are optionally used incombination with procedures that provide additional or synergisticbenefit to the patient. By way of example only, patients are expected tofind therapeutic and/or prophylactic benefit in the methods describedherein, wherein pharmaceutical composition of a compound disclosedherein and/or combinations with other therapeutics are combined withgenetic testing to determine whether that individual is a carrier of amutant gene that is known to be correlated with certain diseases orconditions.

The compounds described herein and combination therapies areadministered before, during or after the occurrence of a disorder, andthe timing of administering the composition containing a compound isvariable. In some embodiments, the compounds are used as a prophylacticand are administered continuously to subjects with a propensity todevelop conditions or diseases in order to prevent the occurrence of thedisorder. In some embodiments, the compounds and compositions areadministered to an individual during or as soon as possible after theonset of the symptoms. In some embodiments, the administration of thecompounds is initiated within the first 48 hours of the onset of thesymptoms, within the first 6 hours of the onset of the symptoms, orwithin 3 hours of the onset of the symptoms. In some embodiments, theinitial administration is via any route practical, such as, for example,an intravenous injection, a bolus injection, infusion over 5 minutes toabout 5 hours, a pill, a capsule, transdermal patch, buccal delivery,and the like, or combination thereof. A compound should be administeredas soon as is practicable after the onset of a disorder is detected orsuspected, and for a length of time necessary for the treatment of thedisease, such as, for example, from about 1 month to about 3 months. Thelength of treatment can vary for each subject, and the length can bedetermined using the known criteria. In some embodiments, the compoundor a formulation containing the compound is administered for at least 2weeks, between about 1 month to about 5 years, or from about 1 month toabout 3 years.

Exemplary Therapeutic Agents for Use in Combination with an IrreversibleInhibitor Compound

In some embodiments, where the subject is suffering from or at risk ofsuffering from a disorder characterized by the presence or developmentof one or more solid tumors, the subjected is treated with anirreversible Btk inhibitor compound in any combination with one or moreother anti-cancer agents. In some embodiments, one or more of theanti-cancer agents are proapoptotic agents. Examples of anti-canceragents include, but are not limited to, any of the following: gossyphol,genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA),bryostatin, tumor necrosis factor-related apoptosis-inducing ligand(TRAIL), 5-aza-2′-deoxycytidine, all trans retinoic acid, doxorubicin,vincristine, etoposide, gemcitabine, imatinib (Gleevec®), geldanamycin,17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol,LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, or PD184352,Taxol™, also referred to as “paclitaxel”, which is a well-knownanti-cancer drug which acts by enhancing and stabilizing microtubuleformation, and analogs of Taxol™, such as Taxotere™. Compounds that havethe basic taxane skeleton as a common structure feature, have also beenshown to have the ability to arrest cells in the G2-M phases due tostabilized microtubules and, in some embodiments, are useful fortreating cancer in combination with the compounds described herein.

Further examples of anti-cancer agents for use in combination with anirreversible Btk inhibitor compound include inhibitors ofmitogen-activated protein kinase signaling, e.g., U0126, PD98059,PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006,wortmannin, or LY294002; Syk inhibitors; mTOR inhibitors; and antibodies(e.g., rituxan).

Other anti-cancer agents for use in combination with an irreversible Btkinhibitor compound include Adriamycin, Dactinomycin, Bleomycin,Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride;acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantroneacetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine;gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride;ifosfamide; iimofosine; interleukin Il (including recombinantinterleukin II, or rlL₂), interferon alfa-2a; interferon alfa-2b;interferon alfa-n1; interferon alfa-n3; interferon beta-1 a; interferongamma-1 b; iproplatin; irinotecan hydrochloride; lanreotide acetate;letrozole; leuprolide acetate; liarozole hydrochloride; lometrexolsodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimi de; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Other anti-cancer agents for use in combination with an irreversible Btkinhibitor compound include: 20-epi-1, 25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron;doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen;ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur;epirubicin; epristeride; estramustine analogue; estrogen agonists;estrogen antagonists; etanidazole; etoposide phosphate; exemestane;fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

Yet other anticancer agents for use in combination with an irreversibleBtk inhibitor compound include alkylating agents, antimetabolites,natural products, or hormones, e.g., nitrogen mustards (e.g.,mechloroethamine, cyclophosphamide, chlorambucil, etc.), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne,ete.), or triazenes (decarbazine, etc.). Examples of antimetabolitesinclude but are not limited to folic acid analog (e.g., methotrexate),or pyrimidine analogs (e.g., Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin).

Examples of natural products useful in combination with an irreversibleBtk inhibitor compound include but are not limited to vinca alkaloids(e.g., vinblastin, vincristine), epipodophyllotoxins (e.g., etoposide),antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g.,L-asparaginase), or biological response modifiers (e.g., interferonalpha).

Examples of alkylating agents for use employed in combination anirreversible Btk inhibitor compound include, but are not limited to,nitrogen mustards (e.g., mechloroethamine, cyclophosphamide,chlorambucil, meiphalan, etc.), ethylenimine and methylmelamines (e.g.,hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan),nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin,etc.), or triazenes (decarbazine, ete.). Examples of antimetabolitesinclude, but are not limited to folic acid analog (e.g., methotrexate),or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine),purine analogs (e.g., mercaptopurine, thioguanine, pentostatin.

Examples of hormones and antagonists useful in combination with anirreversible Btk inhibitor compound include, but are not limited to,adrenocorticosteroids (e.g., prednisone), progestins (e.g.,hydroxyprogesterone caproate, megestrol acetate, medroxyprogesteroneacetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol),antiestrogen (e.g., tamoxifen), androgens (e.g., testosteronepropionate, fluoxymesterone), antiandrogen (e.g., flutamide),gonadotropin releasing hormone analog (e.g., leuprolide). Other agentsfor use in the methods and compositions described herein for thetreatment or prevention of cancer include platinum coordinationcomplexes (e.g., cisplatin, carboblatin), anthracenedione (e.g.,mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazinederivative (e.g., procarbazine), adrenocortical suppressant (e.g.,mitotane, aminoglutethimide).

Examples of anti-cancer agents which act by arresting cells in the G2-Mphases due to stabilized microtubules and which can be used incombination with an irreversible Btk inhibitor compound include withoutlimitation marketed drugs and drugs in development.

Where the subject is suffering from or at risk of suffering from athromboembolic disorder (e.g., stroke), in some embodiments, theindividual is treated with an irreversible Btk inhibitor compound in anycombination with one or more other anti-thromboembolic agents. Examplesof anti-thromboembolic agents include, but are not limited any of thefollowing: thrombolytic agents (e.g., alteplase anistreplase,streptokinase, urokinase, or tissue plasminogen activator), heparin,tinzaparin, warfarin, dabigatran (e.g., dabigatran etexilate), factor Xainhibitors (e.g., fondaparinux, draparinux, rivaroxaban, DX-9065a,otamixaban, LY517717, or YM150), factor VIIa inhibitors, ticlopidine,clopidogrel, CS-747 (prasugrel, LY640315), ximelagatran, or BIBR 1048.

Pharmaceutical Composition/Formulation

Pharmaceutical compositions are formulated in a conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen. Asummary of pharmaceutical compositions described herein is found, forexample, in Remington: The Science and Practice of Pharmacy, NineteenthEd (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

A pharmaceutical composition, as used herein, refers to a mixture of acompound described herein, such as, for example, compounds of any ofFormula (A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6),Formula (I), or Formula (VII), with other chemical components, such ascarriers, stabilizers, diluents, dispersing agents, suspending agents,thickening agents, and/or excipients. The pharmaceutical compositionfacilitates administration of the compound to an organism. In practicingthe methods of treatment or use provided herein, therapeuticallyeffective amounts of compounds described herein are administered in apharmaceutical composition to a mammal having a disorder to be treated.Preferably, the mammal is a human. The compounds can be used singly orin combination with one or more therapeutic agents as components ofmixtures.

The pharmaceutical formulations described herein are administered to anindividual by any suitable administration route, including but notlimited to, oral, parenteral (e.g., intravenous, subcutaneous,intramuscular), intranasal, buccal, topical, rectal, or transdermaladministration routes. The pharmaceutical formulations described hereininclude, but are not limited to, aqueous liquid dispersions,self-emulsifying dispersions, solid solutions, liposomal dispersions,aerosols, solid dosage forms, powders, immediate release formulations,controlled release formulations, fast melt formulations, tablets,capsules, pills, delayed release formulations, extended releaseformulations, pulsatile release formulations, multiparticulateformulations, and mixed immediate and controlled release formulations.

Pharmaceutical compositions including a compound described herein areoptionally manufactured in a conventional manner, such as, by way ofexample only, by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orcompression processes.

The pharmaceutical compositions will include at least one compounddescribed herein, such as, for example, a compound of any of Formula(A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I),or Formula (VII), as an active ingredient in free-acid or free-baseform, or in a pharmaceutically acceptable salt form. In addition, themethods and pharmaceutical compositions described herein include the useof N-oxides, crystalline forms (also known as polymorphs), as well asactive metabolites of these compounds having the same type of activity.In some situations, compounds exist as tautomers. All tautomers areincluded within the scope of the compounds presented herein.Additionally, in some embodiments, the compounds described herein existin unsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. The solvated forms of thecompounds presented herein are also considered to be disclosed herein.

A “carrier” or “carrier materials” includes excipients in pharmaceuticsand is selected on the basis of compatibility with compounds disclosedherein, such as, compounds of any of Formula (A1-A6), Formula (B1-B6),Formula (C1-C6), Formula (D1-D6), Formula (I), or Formula (VII), and therelease profile properties of the desired dosage form. Exemplary carriermaterials include, e.g., binders, suspending agents, disintegrationagents, filling agents, surfactants, solubilizers, stabilizers,lubricants, wetting agents, diluents, and the like. See, e.g.,Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton,Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975;Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms andDrug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

A “measurable serum concentration” or “measurable plasma concentration”describes the blood serum or blood plasma concentration, typicallymeasured in mg, μg, or ng of therapeutic agent per ml, dl, or l of bloodserum, absorbed into the bloodstream after administration. As usedherein, measurable plasma concentrations are typically measured in ng/mlor μg/ml.

“Pharmacodynamics” refers to the factors which determine the biologicresponse observed relative to the concentration of drug at a site ofaction. “Pharmacokinetics” refers to the factors which determine theattainment and maintenance of the appropriate concentration of drug at asite of action.

“Steady state,” as used herein, is when the amount of drug administeredis equal to the amount of drug eliminated within one dosing intervalresulting in a plateau or constant plasma drug exposure.

Dosage Forms

Moreover, the pharmaceutical compositions described herein, whichinclude a compound of any of Formula (A1-A6), Formula (B1-B6), Formula(C1-C6), Formula (D1-D6), Formula (I), or Formula (VII) are formulatedinto any suitable dosage form, including but not limited to, aqueousoral dispersions, liquids, gels, syrups, elixirs, slurries, suspensionsand the like, for oral ingestion by an individual to be treated, solidoral dosage forms, aerosols, controlled release formulations, fast meltformulations, effervescent formulations, lyophilized formulations,tablets, powders, pills, dragees, capsules, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations.

The pharmaceutical solid dosage forms described herein optionallyinclude a compound described herein and one or more pharmaceuticallyacceptable additives such as a compatible carrier, binder, fillingagent, suspending agent, flavoring agent, sweetening agent,disintegrating agent, dispersing agent, surfactant, lubricant, colorant,diluent, solubilizer, moistening agent, plasticizer, stabilizer,penetration enhancer, wetting agent, anti-foaming agent, antioxidant,preservative, or one or more combination thereof. In still otheraspects, using standard coating procedures, such as those described inRemington's Pharmaceutical Sciences, 20th Edition (2000), a film coatingis provided around the formulation of the compound of any of Formula(A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I),or Formula (VII). In one embodiment, some or all of the particles of thecompound of any of Formula (A1-A6), Formula (B1-B6), Formula (C1-C6),Formula (D1-D6), Formula (I), or Formula (VII), are coated. In anotherembodiment, some or all of the particles of the compound of any ofFormula (A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6),Formula (I), or Formula (VII), are microencapsulated. In still anotherembodiment, the particles of the compound of any of Formula (A1-A6),Formula (B1-B6), Formula (C1-C6), Formula (D1-D6), Formula (I), orFormula (VII), are not microencapsulated and are uncoated.

Examples of Methods of Dosing and Treatment Regimens

In some embodiments, the compounds described herein are used in thepreparation of medicaments for the inhibition of Btk or a homologthereof, or for the treatment of diseases or conditions that benefit, atleast in part, from inhibition of Btk or a homolog thereof. In someembodiments, the compounds described herein are used in the preparationof medicaments for the inhibition of HER4 or a homolog thereof, or forthe treatment of diseases or conditions that benefit, at least in part,from inhibition of HER4 or a homolog thereof. In addition, a method fortreating any of the diseases or conditions described herein in anindividual in need of such treatment, involves administration ofpharmaceutical compositions containing at least one compound of any ofFormula (A1-A6), Formula (B1-B6), Formula (C1-C6), Formula (D1-D6),Formula (I), or Formula (VII), described herein, or a pharmaceuticallyacceptable salt, pharmaceutically acceptable N-oxide, pharmaceuticallyactive metabolite, pharmaceutically acceptable prodrug, orpharmaceutically acceptable solvate thereof, in therapeuticallyeffective amounts to said subject.

In some embodiments, the compositions containing the compound(s)described herein are administered for prophylactic and/or therapeutictreatments. In therapeutic applications, the compositions areadministered to an individual already suffering from a disorder, in anamount sufficient to cure or at least partially arrest the symptoms ofthe disorder. Amounts effective for this use will depend on the severityand course of the disorder, previous therapy, the patient's healthstatus, weight, and response to the drugs, and the judgment of thetreating physician.

In prophylactic applications, compositions containing the compoundsdescribed herein are administered to an individual susceptible to orotherwise at risk of a particular disease, disorder. Such an amount isdefined to be a “prophylactically effective amount or dose.” In thisuse, the precise amounts also depend on the patient's state of health,weight, and the like. When used in an individual, effective amounts forthis use will depend on the severity and course of the disease,disorder, previous therapy, the patient's health status and response tothe drugs, and the judgment of the treating physician.

In some embodiments, the irreversible kinase inhibitor is administeredto the patient on a regular basis, e.g., three times a day, two times aday, once a day, every other day or every 3 days. In other embodiments,the irreversible kinase inhibitor is administered to the patient on anintermittent basis, e.g., twice a day followed by once a day followed bythree times a day; or the first two days of every week; or the first,second and third day of a week. In some embodiments, intermittent dosingis as effective as regular dosing. In further or alternativeembodiments, the irreversible kinase inhibitor is administered only whenthe patient exhibits a particular symptom, e.g., the onset of pain, orthe onset of a fever, or the onset of an inflammation, or the onset of askin disorder.

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the compounds may beadministered chronically, that is, for an extended period of time,including throughout the duration of the patient's life in order toameliorate or otherwise control or limit the symptoms of the patient'sdisorder.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds may be givencontinuously; alternatively, the dose of drug being administered may betemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). The length of the drug holiday can varybetween 2 days and 1 year, including by way of example only, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days,180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or365 days. The dose reduction during a drug holiday may be from 10%-100%,including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder isretained. Patients can, however, require intermittent treatment on along-term basis upon any recurrence of symptoms.

The amount of a given agent that will correspond to such an amount willvary depending upon factors such as the particular compound, disorderand its severity, the identity (e.g., weight) of the subject or host inneed of treatment, and is determined according to the particularcircumstances surrounding the case, including, e.g., the specific agentbeing administered, the route of administration, the condition beingtreated, and the subject or host being treated. In general, however,doses employed for adult human treatment will typically be in the rangeof 0.02-5000 mg per day, or from about 1-1500 mg per day. The desireddose may conveniently be presented in a single dose or as divided dosesadministered simultaneously (or over a short period of time) or atappropriate intervals, for example as two, three, four or more sub-dosesper day.

The pharmaceutical composition described herein may be in unit dosageforms suitable for single administration of precise dosages. In unitdosage form, the formulation is divided into unit doses containingappropriate quantities of one or more compound. The unit dosage may bein the form of a package containing discrete quantities of theformulation. Non-limiting examples are packaged tablets or capsules, andpowders in vials or ampoules. Aqueous suspension compositions can bepackaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers can be used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection may be presented in unitdosage form, which include, but are not limited to ampoules, or inmulti-dose containers, with an added preservative.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesmay be altered depending on a number of variables, not limited to theactivity of the compound used, the disorder to be treated, the mode ofadministration, the requirements of the individual subject, the severityof the disorder being treated, and the judgment of the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (thedose therapeutically effective in 50% of the population). The dose ratiobetween the toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio between LD₅₀ and ED₅₀. Compoundsexhibiting high therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with minimal toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

Dosing Strategies to Increase Selectivity

Described herein are irreversible kinase inhibitors that are selectivefor one or more ACKs, including Btk, a Btk homolog, an ACK, HER4, and aBtk kinase cysteine homolog. In some embodiments, the irreversibleinhibitors described herein also bind reversibly to other kinases (someof which, in some embodiments, are also ACKs). As a means of enhancingthe selectivity profile, such inhibitors are formulated (formulationincludes chemical modifications of the inhibitor, use of excipients in apharmaceutical composition, and combinations thereof) such that thepharmacokinetic profile favors enhanced selectivity of the inhibitorsfor an ACK over a non-ACK. By way of example only, an ACK is formulatedto have a short plasma half-life. In other embodiments, an ACK isformulated to have an extended plasma half-life.

For example, as shown in the Examples, Compound 1 and Compound 12 have ashort half-life in vivo. In contrast, Compound 7 and Compound 8 have asignificantly longer in vivo half-life (FIG. 5). Compounds like 1 and 12are predicted to have enhanced kinase selectivity in vivo becauseinhibition will be sustained only for those kinases that areirreversibly inhibited. Further, given that the irreversible kinaseinhibitors described herein have both reversible (in general tonon-ACKs) and irreversible (generally, to ACKs) activities, in vivoproperties of absorption, distribution, metabolism and excretion (ADME)are selected in order to optimize the therapeutic index. Specifically,in some embodiments, rapidly cleared compounds cause only briefinhibition of reversibly inhibited targets while maintaining sustainedinhibition of irreversibly inhibited targets. Depending on the degree towhich sustained inhibition of particular targets results in therapeuticeffects or toxicities, we identify compounds with an optimal combinationof in vitro selectivity profiles and in vivo ADME properties.

In one embodiment are kinase inhibitors that selectively andirreversibly binds to a protein tyrosine kinase selected from Btk, a Btkhomolog, an ACK, HER4, and a Btk kinase cysteine homolog, in which thekinase inhibitor reversibly and non-selectively binds to a multiplicityof protein tyrosine kinases, and further in which the plasma half lifeof the kinase inhibitor is less than about 4 hours. In such anembodiment, the kinase inhibitor selectively and irreversibly binds toat least one of Btk, Jak3, Blk, Bmx, Tec, and Itk. In a furtherembodiment, the kinase inhibitor selectively and irreversibly binds toBtk. In a further embodiment, the kinase inhibitor selectively andirreversibly binds to Jak3. In a further embodiment, the kinaseinhibitor selectively and irreversibly binds to Tec. In a furtherembodiment, the kinase inhibitor selectively and irreversibly binds toBtk and Tec. In a further embodiment, the kinase inhibitor selectivelyand irreversibly binds to Blk. In a further embodiment, the kinaseinhibitor reversibly and non-selectively binds to a multiplicity ofsrc-family protein kinase inhibitors. In a further embodiment, theplasma half life of the kinase inhibitor is less than about 3 hours. Ina further embodiment, the plasma half life of the kinase inhibitor isless than about 2 hours.

In one embodiment are kinase inhibitors that selectively andirreversibly binds to a protein tyrosine kinase selected from Btk, a Btkhomolog, an ACK, HER4, and a Btk kinase cysteine homolog, in which thekinase inhibitor reversibly and non-selectively binds to a multiplicityof protein tyrosine kinases, and further in which the plasma half lifeof the kinase inhibitor is greater than about 12 hours. In such anembodiment, the kinase inhibitor selectively and irreversibly binds toat least one of Btk, Jak3, Blk, Bmx, Tec, and Itk. In a furtherembodiment, the kinase inhibitor selectively and irreversibly binds toBtk. In a further embodiment, the kinase inhibitor selectively andirreversibly binds to Jak3. In a further embodiment, the kinaseinhibitor selectively and irreversibly binds to Tec. In a furtherembodiment, the kinase inhibitor selectively and irreversibly binds toBtk and Tec. In a further embodiment, the kinase inhibitor selectivelyand irreversibly binds to Blk. In a further embodiment, the kinaseinhibitor reversibly and non-selectively binds to a multiplicity ofsrc-family protein kinase inhibitors In a further embodiment, the kinaseinhibitor the plasma half life of the kinase inhibitor is greater thanabout 16 hours.

In one particular embodiment of any of the aforementioned kinaseinhibitors, such kinase inhibitors have the structure of Formula (VII):

wherein:wherein

is a moiety that binds to the active site of a kinase, including atyrosine kinase, further including a Btk kinase cysteine homolog;Y is an optionally substituted group selected from among alkylene,heteroalkylene, arylene, heteroarylene, heterocycloalkylene,cycloalkylene, alkylenearylene, alkyleneheteroarylene,alkylenecycloalkylene, and alkyleneheterocycloalkylene;Z is C(═O), OC(═O), NHC(═O), NCH₃C(═O), C(═S), S(═O)_(x), OS(═O)_(x),NHS(═O)_(x), where x is 1 or 2;R₇ and R₈ are independently selected from among H, unsubstituted C₁-C₄alkyl, substituted C₁-C₄alkyl, unsubstituted C₁-C₄heteroalkyl,substituted C₁-C₄heteroalkyl, unsubstituted C₃-C₆cycloalkyl, substitutedC₃-C₆cycloalkyl, unsubstituted C₂-C₆heterocycloalkyl, and substitutedC₂-C₆heterocycloalkyl; orR₇ and R₈ taken together form a bond;R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted orunsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl, C₁-C₈alkylaminoalkyl,C₁-C₈hydroxyalkylaminoalkyl, substituted or unsubstitutedC₃-C₆cycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted C₂-C₈heterocycloalkyl, substituted or unsubstitutedheteroaryl, C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl),C₁-C₄alkyl(C₃-C₈cycloalkyl), or C₁-C₄alkyl(C₂-C₈heterocycloalkyl); andpharmaceutically active metabolites, or pharmaceutically acceptablesolvates, pharmaceutically acceptable salts, or pharmaceuticallyacceptable prodrugs thereof.

In a further embodiment,

on the kinase inhibitor is a substituted fused biaryl moiety selectedfrom

In a further embodiment of such kinases:

Z is C(═O), NHC(═O), NCH₃C(═O), or S(═O)₂.The kinase inhibitor of Claim 49, wherein:each of R₇ and R₈ is H; orR₇ and R₈ taken together form a bond.

In a further embodiment of such kinases:

R₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted orunsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl, C₁-C₈alkylaminoalkyl,C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl,C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl), C₁-C₄alkyl(C₃-C₈cycloalkyl),or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).

In a further embodiment of such kinases:

Y is a 4-, 5-, 6-, or 7-membered cycloalkylene ring; orY is a 4-, 5-, 6-, or 7-membered heterocycloalkylene ring; orY is a C₁-C₄ alkylene, or 4-, 5-, 6-, or 7-membered heterocycloalkylenering.

In another aspect of such dosing methods are pharmaceutical formulationscomprising any of the aforementioned ACK inhibitors and apharmaceutically acceptable excipient. In some embodiments, suchpharmaceutical formulations are formulated for a route of administrationselected from oral administration, parenteral administration, buccaladministration, nasal administration, topical administration, or rectaladministration. In certain embodiments, the pharmaceutical formulationsare formulated for oral administration.

In another aspect of such dosing methods are methods for treatingrheumatoid arthritis comprising administering to an individual any ofthe aforementioned ACK inhibitors that selectively and irreversiblybinds to Btk and Tec.

In yet another aspect of such dosing strategies are methods forincreasing the selectivity of a test protein tyrosine kinase inhibitorthat irreversibly and selectively binds to at least one protein kinaseinhibitor selected from Btk, a Btk homolog, a Btk kinase cysteinehomolog, an ACK, or HER4, in which the test protein tyrosine kinaseinhibitor is chemically modified to decrease the plasma half life toless than about 4 hours. In some embodiments, the test protein tyrosinekinase inhibitor is chemically modified to decrease the plasma half lifeto less than about 3 hours.

In further embodiments, the test protein tyrosine kinase inhibitor hasthe structure of Formula (VII):

wherein

is a moiety that binds to the active site of a kinase, including atyrosine kinase, further including a Btk kinase cysteine homolog;Y is an optionally substituted group selected from among alkylene,heteroalkylene, arylene, heteroarylene, heterocycloalkylene,cycloalkylene, alkylenearylene, alkyleneheteroarylene,alkylenecycloalkylene, and alkyleneheterocycloalkylene;Z is C(═O), OC(═O), NHC(═O), NCH₃C(═O), C(═S), S(═O)_(x), OS(═O)_(x),NHS(═O)_(x), where x is 1 or 2;R₇ and R₈ are independently selected from among H, unsubstituted C₁-C₄alkyl, substituted C₁-C₄alkyl, unsubstituted C₁-C₄heteroalkyl,substituted C₁-C₄heteroalkyl, unsubstituted C₃-C₆cycloalkyl, substitutedC₃-C₆cycloalkyl, unsubstituted C₂-C₆heterocycloalkyl, and substitutedC₂-C₆heterocycloalkyl; orR₇ and R₈ taken together form a bond; andR₆ is H, substituted or unsubstituted C₁-C₄alkyl, substituted orunsubstituted C₁-C₄heteroalkyl, C₁-C₆alkoxyalkyl, C₁-C₈alkylaminoalkyl,C₁-C₈hydroxyalkylaminoalkyl, C₁-C₈alkoxyalkylaminoalkyl, substituted orunsubstituted C₃-C₆cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted C₂-C₈heterocycloalkyl, substituted orunsubstituted heteroaryl, C₁-C₄alkyl(aryl), C₁-C₄alkyl(heteroaryl),C₁-C₄alkyl(C₃-C₈cycloalkyl), or C₁-C₄alkyl(C₂-C₈heterocycloalkyl).

In a further embodiment, the test protein tyrosine kinase inhibitornon-selectively and reversibly binds to a multiplicity of src-familyprotein tyrosine kinases.

In a further aspect of such dosing strategies are methods for treating adisorder characterized by the presence or development of one or moresolid tumors comprising administering to an individual in need apharmaceutical composition of any of the aforementioned ACK inhibitors.For example, as presented in the Examples, brief exposure to Compound 1in vitro is sufficient to inhibit B cell activation in normal human Bcells. This protocol mimics the predicted exposure of cells to Compound1 in vivo and demonstrates that inhibition of B cells is sustaineddespite washing out of Compound 1.

Kits/Articles of Manufacture

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. In some embodiments,such kits include a carrier, package, or container that iscompartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) including one of theseparate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers can be formed from a variety of materials such asglass or plastic.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical productsinclude, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials,containers, syringes, bottles, and any packaging material suitable for aselected formulation and intended mode of administration and treatment.A wide array of formulations of the compounds and compositions providedherein are contemplated as are a variety of treatments for any disorderthat benefit by inhibition of Btk, or in which Btk is a mediator orcontributor to the symptoms or cause.

For example, the container(s) include one or more compounds describedherein, optionally in a composition or in combination with another agentas disclosed herein. The container(s) optionally have a sterile accessport (for example the container is an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). Such kitsoptionally comprising a compound with an identifying description orlabel or instructions relating to its use in the methods describedherein.

A kit will typically include one or more additional containers, eachwith one or more of various materials (such as reagents, optionally inconcentrated form, and/or devices) desirable from a commercial and userstandpoint for use of a compound described herein. Non-limiting examplesof such materials include, but not limited to, buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use. A set of instructions will alsotypically be included.

In some embodiments, a label is on or associated with the container. Alabel can be on a container when letters, numbers or other charactersforming the label are attached, molded or etched into the containeritself; a label can be associated with a container when it is presentwithin a receptacle or carrier that also holds the container, e.g., as apackage insert. A label can be used to indicate that the contents are tobe used for a specific therapeutic application. The label can alsoindicate directions for use of the contents, such as in the methodsdescribed herein.

In certain embodiments, the pharmaceutical compositions can be presentedin a pack or dispenser device which can contain one or more unit dosageforms containing a compound provided herein. The pack can for examplecontain metal or plastic foil, such as a blister pack. The pack ordispenser device can be accompanied by instructions for administration.The pack or dispenser can also be accompanied with a notice associatedwith the container in form prescribed by a governmental agencyregulating the manufacture, use, or sale of pharmaceuticals, whichnotice is reflective of approval by the agency of the form of the drugfor human or veterinary administration. Such notice, for example, can bethe labeling approved by the U.S. Food and Drug Administration forprescription drugs, or the approved product insert. Compositionscontaining a compound provided herein formulated in a compatiblepharmaceutical carrier can also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition.

EXAMPLES

The following specific and non-limiting examples are to be construed asmerely illustrative, and do not limit the present disclosure in any waywhatsoever.

Synthesis of Compounds Example 1: Preparation of4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine (Intermediate2)

4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine (Intermediate2) is prepared as disclosed in International Patent Publication No. WO01/019829. Briefly, 4-phenoxybenzoic acid (48 g) is added to thionylchloride (100 mL) and heated under gentle reflux for 1 hour. Thionylchloride is removed by distillation, the residual oil dissolved intoluene and volatile material removed at 80° C./20 mbar. The resultingacid chloride is dissolved in toluene (200 mL) and tetrahydrofuran (35mL). Malononitrile (14.8 g) is added and the solution and stirred at−10° C. while adding diisopropylethylethylamine (57.9 g) in toluene (150mL), while maintaining the temperature below 0° C. After 1 hour at 0°C., the mixture is stirred at 20° C. overnight. Amine hydrochloride isremoved by filtration and the filtrate evaporated in vacuo. The residueis taken up in ethyl acetate and washed with 1.25 M sulphuric acid, thenwith brine and dried over sodium sulfate. Evaporation of the solventsgives a semisolid residue which is treated with a little ethyl acetateto give 4.1 g of 1,1-dicyano-2-hydroxy-2-(4-phenoxyphenyl)ethene as awhite solid (m.p. 160-162° C.). The filtrate on evaporation gives 56.58(96%) of 1,1-dicyano-2-hydroxy-2-(4-phenoxyphenyl)ethene as a grey-brownsolid, which is sufficiently pure for further use.

1,1-Dicyano-2-hydroxy-2-(4-phenoxyphenyl)ethene (56.5 g) in acetonitrile(780 mL) and methanol (85 mL) is stirred under nitrogen at 0° C. whileadding diisopropylethylamine (52.5 mL) followed by 2Mtrimethylsilyldiazomethane (150 mL) in THF. The reaction is stirred for2 days at 20° C., and then 2 g of silica is added (for chromatography).The brown-red solution is evaporated in vacuo, the residue dissolved inethyl acetate and washed well with water then brine, dried andevaporated. The residue is extracted with diethyl ether (3×250 mL),decanting from insoluble oil. Evaporation of the ether extracts gives22.5 g of 1,1-dicyano-2-methoxy-2-(4-phenoxyphenyl)ethene as a paleorange solid. The insoluble oil is purified by flash chromatography togive 15.0 g of a red-orange oil.1,1-Dicyano-2-methoxy-2-(4-phenoxyphenyl)ethene (22.5 g) and1,1-dicyano-2-methoxy-2-(4-phenoxyphenyl)ethene oil (15 g) are treatedwith a solution of hydrazine hydrate (18 mL) in ethanol (25 mL) andheated on the steambath for 1 hour. Ethanol (15 mL) is added followed bywater (10 mL). The precipitated solid is collected and washed withethanol:water (4:1) and then dried in air to give3-amino-4-cyano-5-(4-phenoxyphenyl)pyrazole as a pale orange solid.

3-Amino-4-cyano-5-(4-phenoxyphenyl)pyrazole (29.5 g) is suspended informamide (300 mL) and heated under nitrogen at 180° C. for 4 hours. Thereaction mixture is cooled to 30° C. and water (300 mL) is added. Thesolid is collected, washed well with water, then with methanol and driedin air to give of4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine.

Example 2: Synthesis of1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(Compound 13)

Synthesis of Compound 13; a) triphenylphosphine (TPP), diisopropyldiazodicarboxylate (DIAD), tetrahydrofuran (THF); b) TFA/CH₂Cl₂; thenacryloyl chloride, diisopropylethylamine (DIPEA), tetrahydrofuran (THF).

Compounds described herein were synthesized by following the stepsoultined in Scheme 1. A detailed illustrative example of the reactionconditions shown in Scheme 1 is described for the synthesis of1-((R)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)prop-2-en-1-one(Compound 13).

0.5 g of 4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine and0.65 g of triphenylphosphine (TPP) were mixed together with 15 mL oftetrahydrofuran (THF). (R)-tert-butyl2-(hydroxymethyl)pyrrolidine-1-carboxylate (0.5 g; 1.5 equivalents) wasadded to the mixture followed by the addition of diisopropyldiazodicarboxylate (0.5 mL). The reaction mixture was stirred at roomtemperature for 4 hr. The reaction mixture was concentrated and purifiedby flash chromatography (acetone/CH₂Cl₂=1/1) to give intermediate 3(1.49 g).

Intermediate 3 (1.49 g) was treated with 4 mL of TFA and 5 mL of CH₂Cl₂and stirred overnight at room temperature and then concentrated todryness. The residue was dissolved in ethyl acetate (100 mL) and thenwashed with dilute aq. NaHCO₃ (100 mL). The ethyl acetate layer wasdried (MgSO₄), filtered and concentrated to ˜20 mL and then 4.0 MHCl\dioxane (1 mL) was added and a yellow precipitate formed. The solidwas collected by filtration and washed with ethyl acetate (20 mL). Thesolid was suspended in ethyl acetate (100 mL) and again washed withdilute aq. NaHCO₃ (100 mL). The ethyl acetate was dried (MgSO₄),filtered and concentrated to provide 0.43 g of a light yellow solid. Thesolid (0.14 g, 0.36 mmol) was stirred in THF (3 mL) and TEA (015 mL, 1.1mmol) was added, followed by cooling the reaction with an ice bath for30 min, then acryl chloride (30 μL, 0.36 mmol) added and the reactionwas stirred for 2 hr. The reaction mixture was diluted with ethylacetate (75 mL) and washed with dilute aq. NaHCO₃ (100 mL). The organiclayer was dried (MgSO₄), filtered and concentrated. Flash chromatography(with CH₂Cl₂/MeOH=20/1) gave 90 mg of compound 4 as a white solid. EM(calc)=440.2; MS (M+1): 441.2.

Example 3: Synthesis of1-((S)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)prop-2-en-1-one(Compound 14)

The synthesis of Compound 14 was accomplished using a procedureanalogous to that described in Example 2. EM (calc.): 440.2; MS (M+1H):441.2.

Example 4: Synthesis ofN-((1r,4r))-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)acrylamide

The synthesis of this compound was accomplished using a procedureanalogous to that described for Example 2 EM (calc.): 454.21; MS (M+1):455.2.

Example 5: Synthesis ofN-(2-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-methylacrylamide(Compound 19)

The synthesis of this compound was accomplished using a procedureanalogous to that described for Example 2. EM (calc.): 414.18; MS(M+1H): 415.2.

Example 6: Synthesis ofN-(2-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)acrylamide(Compound 23)

The synthesis of this compound was accomplished using a procedureanalogous to that described for Example 2. EM (calc.): 400.16; MS(M+1H): 401.2.

Example 7: Synthesis of1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)but-2-yn-1-onee (Compound 17)

The synthesis of this compound was accomplished using a procedureanalogous to that described for Example 2. EM (calc.): 452.2; MS (M+1H):453.2.

Example 8: Synthesis of1-((R)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)but-2-yn-1-one(Compound 15)

The synthesis of this compound was accomplished using a procedureanalogous to that described for Example 2. EM (calc.): 452.2; MS (M+1H):453.2.

Example 9: Synthesis of(E)-1-((R)-2-((4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidin-1-yl)-4-(dimethylamino)but-2-en-1-one(Compound 11)

The synthesis of this compound was accomplished using a procedureanalogous to that described for Example 2. EM (calc.): 497.25; MS(M+1H): 498.2.

Therapeutic Uses of Inhibitor Compounds Example 1: Inhibition of LyphomaTumor Cell Growth

Compound 1 inhibits lymphoma tumor cell growth. A variety of lymphomacell lines were incubated with a range of concentrations of Compound 1to determine the GI50, the concentration that results in 50% decrease incell proliferation (FIG. 1A). Compound 1 inhibits tumor growth in DOHH2and DLCL2 xenograft models (FIGS. 1B and 1C).

For in vitro cell proliferation assays, cells were seeded in 96-wellplates in standard growth media (in most cases RPMI+10% fetal calfserum) and Compound 1 was added in a 9-point dilution series rangingfrom 10 uM to 0.04 uM with DMSO at 0.1% final concentration in allwells. After 72 hours, cell number was measured using Alamar Blue usingmanufacturer's protocol. A dilution series of untreated cells was run inparallel to verify that the Alamar Blue assay reliably reflected cellnumber and that growth conditions were not limiting. The GI50, theconcentration that results in a 50% decrease in cell number, wascalculated using Calcusyn to fit the dose-response curve. GI50 valueswere confirmed in two or more separate experiments for each cell line.

For in vivo lymphoma xenograft studies, 5E6 DOHH2 or DLCL2 cells in 50%matrigel were implanted subcutaneously in SCID mice and dosed orallywith Compound 1 beginning when tumor size reached 100 mm2.

Example 2: Inhibition of Collagen-Induced Arthritis in a Mouse

Compound 1 inhibits collagen-induced arthritis in the mouse. MaleDBA/1OlaHsd mice were injected intradermally with 150 microliters of 2mg/mL Type II collagen in Freund's complete adjuvant with supplementalM. tuberculosis, 4 mg/mL and boosted with the same injection 21 dayslater. After paw inflammation was established, animals were randomizedand Compound for vehicle was dosed orally once per day starting atday 1. Paw inflammation was scored from 0-5 and averaged across all pawsfrom all animals for each group in the study. Compound 1 at 12.5 mg/kgand 50 mg/kg regressed inflammation through the end of the study (day11) while 3.125 mg/kg significantly reduced the increase in pawinflammation (FIG. 2). Dexamethasone was included as a positive control.

In another study, Compound 1 was dosed at 12.5 mg/kg to such mice over:(a) each day of an 11-day period; (b) days 1, 2, and 3 of an 11-dayperiod; or (c) days 9, 10, and 11 of an 11-day period. Intermittentdosing reduced the increase in paw inflammation. In addition, Compound 9was dosed to such mice at a level of 12.5 mg/kg or 50 mg/kg each day ofan 11-day period. Compound 9 reduced the increase in paw inflammation.

Example 3: Inhibition of Lupus in a Mouse Model

Compound 1 inhibits disease progression in the mouse MRL/lpr model oflupus. Compound 1 at 3.125 mg/kg, 12.5 mg/kg, and 50 mg/kg significantlyreduced proteinuria, indicating amelioration of the progressiveautoimmune renal failure seen in this mouse strain (FIG. 3). MRL/lprmice (Jax strain 000485) were dosed orally once per day from 12 weeks ofage until 20 weeks of age and urine protein levels were measured weeklyusing Clinitech Multistick dipstick.

Example 4: Inhibition of Mast Cell Degranulation

Compound 1 inhibits mast cell degranulation in a mouse passive cutaneousanaphylaxis model. Increasing doses of Compound 1 significantly decreasethe amount of Evans Blue release, indicating decreased mast cellactivation and vascular permeabilization. (FIG. 4)

Mice were sensitized with an intradermal injection of monoclonalanti-DNP-IgE in the back. 23 hours later they received a single oraldose of Compound 1 or vehicle. After one hour, animals were challengedwith an intravenous injection of DNP-BSA and Evans Blue dye. Mast celldegranulation leads to vascular permeability and the distribution of thedye into the skin of the back. The area of extravasation after 1 hour ismeasured.

Example 5: Pharmaceutical Compositions

The compositions described below are presented with a compound ofFormula (A1-A6) for illustrative purposes; any of the compounds of anyof Formulas (A1-A6), (B1-B6), (C1-C6), or (D1-D6) are optionally used insuch pharmaceutical compositions.

Example 5a: Parenteral Composition

To prepare a parenteral pharmaceutical composition suitable foradministration by injection, 100 mg of a water-soluble salt of acompound of Formula (A1-A6) is dissolved in DMSO and then mixed with 10mL of 0.9% sterile saline. The mixture is incorporated into a dosageunit form suitable for administration by injection.

Example 5b: Oral Composition

To prepare a pharmaceutical composition for oral delivery, 100 mg of acompound of Formula (A1-A6) is mixed with 750 mg of starch. The mixtureis incorporated into an oral dosage unit for, such as a hard gelatincapsule, which is suitable for oral administration.

Example 5c: Sublingual (Hard Lozenge) Composition

To prepare a pharmaceutical composition for buccal delivery, such as ahard lozenge, mix 100 mg of a compound of Formula (A1-A6), with 420 mgof powdered sugar mixed, with 1.6 mL of light corn syrup, 2.4 mLdistilled water, and 0.42 mL mint extract. The mixture is gently blendedand poured into a mold to form a lozenge suitable for buccaladministration.

Example 5d: Inhalation Composition

To prepare a pharmaceutical composition for inhalation delivery, 20 mgof a compound of Formula (A1-A6) is mixed with 50 mg of anhydrous citricacid and 100 mL of 0.9% sodium chloride solution. The mixture isincorporated into an inhalation delivery unit, such as a nebulizer,which is suitable for inhalation administration.

Example 5e: Rectal Gel Composition

To prepare a pharmaceutical composition for rectal delivery, 100 mg of acompound of Formula (A1-A6) is mixed with 2.5 g of methylcellulose (1500mPa), 100 mg of methylparaben, 5 g of glycerin and 100 mL of purifiedwater. The resulting gel mixture is then incorporated into rectaldelivery units, such as syringes, which are suitable for rectaladministration.

Example 5f: Topical Gel Composition

To prepare a pharmaceutical topical gel composition, 100 mg of acompound of Formula (A1-A6) is mixed with 1.75 g of hydroxypropylcellulose, 10 mL of propylene glycol, 10 mL of isopropyl myristate and100 mL of purified alcohol USP. The resulting gel mixture is thenincorporated into containers, such as tubes, which are suitable fortopical administration.

Example 5g: Ophthalmic Solution Composition

To prepare a pharmaceutical ophthalmic solution composition, 100 mg of acompound of Formula (A1-A6) is mixed with 0.9 g of NaCl in 100 mL ofpurified water and filtered using a 0.2 micron filter. The resultingisotonic solution is then incorporated into ophthalmic delivery units,such as eye drop containers, which are suitable for ophthalmicadministration.

Example 6: Levels of Tonic BCR Signaling Predict Response to Compound 1

To identify biomarkers that correlate with response to Compound 1,phosphorylation events in the BCR signal transduction pathway wereinvestigated. A panel of phospho-specific antibodies that recognizeactivating phosphorylation sites on Syk, Btk, BLNK, PLC-g1, PLC-g2, ERK,and AKT were used and tested the effects of Compound 4 on both basalphosphorylation and phosphorylation following BCR stimulation driven byanti-IgM or anti-IgG cross-linking. We examined phosphorylation patternsin both a Compound 1 sensitive cell line (DOHH2) and a Compound 1resistant cell line (Ramos).

Compound 1 inhibits most BCR-stimulus induced phosphorylation eventswith similar potency in both cell lines. However, when we examined basalphosphorylation levels, we found higher basal phosphorylation in DOHH2compared to Ramos, with phospho-ERK in particular indicating higherlevels of basal or tonic signaling in DOHH2. Furthermore, Compound 4significantly decreased pERK levels in unstimulated DOHH2 cells (IC50<10nM), but not in Ramos cells.

A panel of nine Btk expressing B cell lymphoma cell lines was screenedfor basal pERK levels. Seven lines expressed significantly higher levelsof basal pERK, and of these, 5 were sensitive to Compound 1 (GI50<1.3uM), while the two cell lines with low pERK levels were resistant toCompound 1. This data shows that tonic BCR signaling contributes to thesurvival of a subset of lymphoma cell lines, and that inhibition of thissignaling by Compound 4 is correlated with induction of apoptosis.

Two additional experiments demonstrate that sensitivity to Compound 1 iscorrelated with high levels of pERK. First 1 uM of Compound 4 reducesexpression of the known ERK transcriptional target Egr-1 within 1 hr,with maximal downregulation (10-fold) achieved by 4 hr. Second, in thelymphoma cell line WSU-DLCL2, BCR cross-linking by anti-IgG (30 ug/ml)overcomes inhibition of pERK by Compound 4, showing that strong BCRstimulus activates parallel pathways to pERK that do not require Btk.BCR stimulus also rescues WSU-DLCL2 from Compound 1 inducedcytotoxicity, further confirming that inhibition of pERK is correlatedwith apoptosis induction by Compound 1. Taken together these data showhigh levels of pERK or ERK transcriptional targets such as Egr-1 serveas useful markers for lymphomas in which tonic BCR signaling iscontributing to cell survival and that these lymphomas are particularlysensitive to BCR pathway inhibitors such as Compound 1.

Kinase Inhibitor Discovery Platform and Pulse Dosing Example 1: Designof an Inhibitor

Because the ATP binding sites of the >500 kinases in the human genomeare highly conserved, it has proven difficult to engineer selectivityfor individual kinases using conventional reversible binding inhibitors.For our highly selective BTK inhibitor Compound 1, we engineered anelectrophilic center capable of irreversibly inactivating the targetenzyme, BTK. The approach employed structure based design to achieve ahigh degree of potency and selectivity by (1) fitting the core scaffoldinto the active site ATP binding pocket of kinase enzymes, and (2)forming a covalent bond with Cysteine-481 located in BTK. The uniquechemistry required for covalent bond formation involves an electrophilicmoiety that acts as a Michael acceptor, which bonds with a nucleophile(such as Cys-481) present in a precise location within the active site.

Example 2: Inhibitor Screening Approach

By way of example only, a panel of 50-100 Cys-targeting kinaseinhibitors is generated. The molecular orientation and positioning ofthe electrophilic group in these inhibitors in relation to the Cysteineresidue will affect the potency and selectivity of a given inhibitor.Each inhibitor will then be profiled for kinetics of kinase inhibition(KO for each of the ten Cys-containing kinases, effect on tumor cellproliferation (GI₅₀), effect on relevant off-targets (hERG, CYPs),drug-like characteristics (solubility, c log P) and ability to blocklabeling by the active site probe. This panel of diverse inhibitors arethen be used in cell assays (for example, inhibition of tumor growth) toscreen for a phenotype of interest. With the phenotype, theidentification of additional inhibited kinases is determined using theactive site probe and mass spectrometry.

Example 3: Inhibition of a Panel of Kinases for Compound 1 and Compound9

In another example, the linker and Michael acceptor moiety of Compound 1was modified to provide Compound 9 which has a different selectivitypattern. Table 1 is a table showing the degree of inhibition of a panelof kinases for two example compounds. IC₅₀s were determined using the invitro HotSpot kinase assay (purified enzymes, ³³P-ATP, an appropriatesubstrate and 1 uM ATP.) Compared to Compound 1, Compound 9 has similarpotency toward Btk, but significantly less potency toward JAK-3, ITK,and EGFR and significantly more potency toward the src-family kinaseslck, c-src, FGR, Fyn, Hck, and Lyn and Yes. Thus, subtle modificationsin the linker moiety and the Michael acceptor moiety are important forthe design of selective ACK inhibitors.

TABLE 1 Compound 1 Compound 9 Kinase IC50 (nM) IC50 (nM) BTK 0.5 1.0 ITK11.7 909.9 Bmx/ETK 0.8 1.1 TEC 77.8 108.0 EFGR 0.5 20.6 HER4 9.4 1536.0HER4 0.1 3.2 LCK 2.0 1.0 BLK 0.5 0.2 C-src 262.6 14.3 FGR 2.3 0.4 Fyn95.6 7.1 HCK 3.7 1.0 Lyn 16.2 1.2 YES 6.5 0.8 ABL 86.1 32.3 Brk 3.3 3.3CSK 2.2 2.4 FER 8,070.0 3,346.0 JAK3 10.4 8,278.0 SYK >10,000 >10,000

Example 4: Modification of Linker and Michael Acceptor Moieties and InVitro Inhibitory Activity

In this example, compounds are selected based on in vitrocharacteristics to optimize for potency of inhibition of particularkinases and degree of covalent binding to off-target cysteines such asglutathione. For example, in Table 2, Compound 9 and Compound 12 bothinhibit Btk with a similar potency as Compound 1, but they are bothsignificantly less potent inhibitors of EGFR, ITK, and JAK-3. As anotherexample, Compound 11 is similar to Compound 1 for inhibition of Btk butdoes not bind glutathione as readily.

A calculated value (e.g (1/Btk IC₅₀)/Glutathione conjugation rate) asshown in the Table 2) is used to compare compounds for their ratiobetween potency at inhibiting their target and their non-specificbinding to other SH groups, such as those in glutathione. As shown inTable 2, this calculated value is 4.7 for Compound 1 and for 239.6 forCompound 11. Calculated ratios such as these are used to quantitativelycompare different compounds and select compounds for further study.

Example 4a: Enzyme Inhibition

For enzyme inhibition assays, compounds were tested in range of tenconcentrations from 10 uM to 0.0005 uM using purified enzymes and theHotspot kinase assay. Reaction conditions were 1 uM ATP, one hourincubation with inhibitor, and kinase activity detected using 33-ATPphosphorylation of an appropriately selected peptide substrate.Dose-response curves were fit using Prism, and the IC₅₀, theconcentration at which enzyme inhibition is 50% of maximal inhibition,was determined. See Table 2.

Example 4b: Glutathione Binding Assays

For the glutathione binding assays, 5 mM glutathione, 10 μM Btkinhibitor in DMSO (10 μL) and 6 equivalents of N′N′ Diisopropyl ethylamine were combined in 1 mL potassium phosphate buffer. The mixture wasincubated for 0, 15, 60 minutes at room temperature and the reaction wasstopped with 10 equivalents of formic acid. 50 μL of each reactionmixture was injected on HPLC (Mobil Phase A: 0.2% formic acid in water,Mobile Phase B: 0.2% formic acid in acetonitrile, HPLC Column: MetasilBasic 3μ, 150×4.6 mm, 10% B, Gradient: 10% to 90% B, Detection: UV/Vis260 nM). Rate of reaction was reported as nmole GSH conjugate conversionper minute from the normalized ratio for area under the curve from HPLCchromatograms for both GSH conjugate and the parent.

Example 4c: Cell Proliferation Assay

Analogs are generated that are Btk inhibitors and that are cytotoxic tothe lymphoma cell line DOHH2. See Table 2. For the DOHH2 cellproliferation assay, cells were seeded in 96-well plates in standardgrowth media (RPMI+10% fetal calf serum) and compounds were added in a9-point dilution series ranging from 10 uM to 0.04 uM with DMSO at 0.1%final concentration in all wells. After 72 hours, cell number wasmeasured using Alamar Blue using manufacturer's protocol. A dilutionseries of untreated cells was run in parallel to verify that the AlamarBlue assay reliably reflected cell number and that growth conditionswere not limiting. The GI₅₀, the concentration that results in a 50%decrease in cell number, was calculated using Calcusyn to fit thedose-response curve.

TABLE 2 Glutathione (1/BTK DOH BTK ITK EGFR LCK JAK3 Conj IC₅₀)/ H2Compound IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ Rate Glutathione GI₅₀ # Structure (nM)(nM) (nM) (nM) (nM) (nmol/min) Rate (μM)  1

0.5 11.7 0.5 2.0 10.4 0.398 4.7 0.1  2

1.1 48.8 0.32  3

21 74.5  4

22.2 487.6  5

5.6 326.0 0.004 44.5  6

3.1 60.9 0.39 0.8  7

6.3 6,123 268.7 2.6 >10,000 0.01 15.9 0.317  8

1.4 83.4  9

1.0 909.9 20.6 1.0 8278.0 0.011 10

1.31 1954 44.5 0.88 >10,000 <0.03 11

0.92 6891 18.85 2.43 >10,000 0.004525 239.6 >10 12

1.33 14290 698.3 5.97 >10,000 0.004361 172.2 >10 13

0.67 3013 18.75 1.56 12980 0.24 14

0.39 592.3 2.298 9.24 1456 0.37 15

4.16 21100 289.4 5.90 >10,000 0.59 16

3.14 >10,000 2807 3.82 >10,000 0.21 17

2.00 2333 435.3 2.07 >10,000 0.0243 20.6 0.21 18

1.38 2536 22.53 0.76 >10,000 <0.03 19

1.58 534.6 28.22 6.62 5997 0.69 20

4.07 7993 303.60 98.59 >10,000 0.39 21

4.15 >10,000 6238.00 1346 >10,000 1.53 22

1.57 3691 156.30 22.12 >10,000 0.014 45.4 <10.04 23

0.32 830 70.49 208.00 3306.00 0.11 24

0.89 476 383.70 235.40 9077.00 0.44 25

3.48 >10,000 272.90 25.81 >10,000 0.05

Example 5: Kinase Inhibitor Selectivity Predicted by Dosing

Compound 1 and Compound 12 have a short half-life in vivo. In contrast,Compound 7 and Compound 8 have a significantly longer in vivo half-life(FIG. 5). Compounds like 1 and 12 are predicted to have enhanced kinaseselectivity in vivo because inhibition will be sustained only for thosekinases that are irreversibly inhibited.

Male jugular vein cannulated rats were administered a single dose of alltest compounds at 8 mg/kg each, in combination by oral gavage. Dosevolumes were adjusted based on body weight data collected immediatelyprior to dosing. Blood samples were collected at 0.0833 (5 minutes),0.333 (20 minutes), 1, 3, 6, 9, and 24 hours post-dosing from orallydosed rats. The samples were collected into plasma separator Microtainertubes with anticoagulant (lithium heparin). Plasma samples were preparedby centrifugation (5 min at 5000×g), and at least 100 μL weretransferred to storage tubes and stored frozen at −80° C. Plasma sampleswere thawed and 75 uL aliquots were transferred to centrifuge tubes towhich 10 μL aliquots of internal standard solution (1 μg/mL) were added.The samples were not diluted with blank plasma prior to furtherprocessing. Soluble proteins were precipitated by the addition of 200 μLof acetonitrile, followed by centrifugation (20 min at 16,000×g). Thesamples were evaporated to dryness and reconstituted in 200 μL of watercontaining 0.2% formic acid and 10% methanol. All samples were loadedonto an autosampler maintained at 6° C. and evaluated for concentrationsof test compounds using LC-MS/MS.

Example 6: B Cell Inhibition

Brief exposure to Compound 1 in vitro is sufficient to inhibit B cellactivation in normal human B cells (FIG. 6). This protocol mimics thepredicted exposure of cells to Compound 1 in vivo and demonstrates thatinhibition of B cells is sustained despite washing out of Compound 1.

B cells were purified from blood from healthy donors by negativeselecting using the RosetteSep Human B cell enrichment cocktail. Cellswere plated in growth media (10% RPMI+10% fetal calf serum) andindicated concentrations of Compound 1 were added. After incubation for1 hour at 37° C., cells were washed three times using an 8-fold dilutionin growth media for each wash. Cells were then stimulated with 10 ug/mlof IgM F(ab′)2 for 18 hours at 37° C. Cells were then stained withanti-CD69-PE antibody and analyzed by flow cytometry using standardconditions.

Example 7: Optimizing the Therapeutic Index of Kinase Inhibitors

Given that kinase inhibitors described above will have both reversibleand irreversible activities, we select their in vivo properties ofabsorption, distribution, metabolism and excretion (ADME) in order tooptimize the therapeutic index. Specifically, rapidly cleared compoundsare expected to cause only brief inhibition of reversibly inhibitedtargets while maintaining sustained inhibition of irreversibly inhibitedtargets. Depending on the degree to which sustained inhibition ofparticular targets results in therapeutic effects or toxicities, weidentify compounds with an optimal combination of in vitro selectivityprofiles and in vivo ADME properties.

Example 8: Administration of Btk Inhibitor to a Mouse Model for ColonCancer

Under the skin of a mouse is implanted a colon cancer xenograft. On adaily basis, administration of Compound 1 is effected by intravenousadministration at a level of 1 microgram per gram of mouse weight. Thesize of the tumor xenograft is also monitored daily. Success isdetermined by survival of the mouse for a period of time statisticallylonger than survival of a mouse adiminstered with vehicle on the samedosing schedule.

Example 9: Pancreatic Cancer Clinical Trial Length of Study

8 months [length of time from FPV to LPV]

Objectives

The primary objective of this study will be to determine the objectiveresponse rate (ORR) for Btk inhibitors when administered every 2 weeksto patients with adenocarcinoma of the pancreas. The secondaryobjectives of this study will be to measure time-to-event variablesincluding: time to objective tumor response for responding patients(TtOR), duration of response for responding patients, time to treatmentfailure (TtTF), time to progressive disease (TtPD), progression-freesurvival (PFS), overall survival (OS); the toxicities of therapy.

Study Design

The study will be a multi-center, double-blind, randomized,placebo-controlled Phase 2 study. Tumor assessments will be repeatedevery 4 cycles (approximately 8 weeks). Patients will receive studytherapy for 12 treatments, or until tumor progression was documented,unacceptable toxicity was experienced, the patient withdrew consent, orthe patient is unable to fulfill the responsibilities of studyparticipation as determined by the treating physician or the qualifiedinvestigator. After study discontinuation, patients who have notprogressed will have tumor assessments performed approximately every 8weeks until disease progression. Once patients have disease progression,patients will enter a post-study follow-up period, and will be followedevery 12 weeks for 24 months for overall survival. Patients will also befollowed for ongoing or any new toxicities.

Diagnosis and Main Criteria for Inclusion:

Male and females ≥18 years of age will be eligible for this study ifthey are diagnosed with adenocarcinoma of the pancrease. Patients musthave had as their initial presentation pancreatic metastasis withoutevidence of pulmonary metastasis.

Main inclusion criteria will include: histologically provenadenocarcinoma; performance Status of 0 or 1 on the Eastern CooperativeOncology Group (ECOG) scale; a complete history and physical, chestx-ray, CT scan of abdomen and pelvis; barium enema, or colonoscopy.Patients with pain will be requires to have had their pain stabilizedfor 1 week prior to commencing therapy. Patients requiring opioids forpain control will be required to have been on a fixed analgesic regimenaimed to provide adequate pain control with no more than threebreakthrough (supplemental) doses of analgesics per day to control pain.Patients will be requires to demonstrate adequate bone marrow reserve(i.e. Neutrophil count≥1.5×109 cells/L; Platelets≥100×109 cells/L).Patients will be required to have negative tumor markers foralpha-fetoprotein (AFP) and monoclonal antichorionic gonadotropin(β-subunit) (βHCG). Patients will be required to demonstrate at leastone unidimensionally measurable lesion, meeting Response EvaluationCriteria in Solid Tumors (RECIST). Patient will also be required to havean estimated life expectancy of at least 12 weeks.

Main exclusion criteria will include: prior chemotherapy; pregnancy orbreastfeeding; inability or unwillingness to take folic acid, vitaminB12 supplementation, or dexamethasone.

Study Drug, Dose, and Mode of Administration

Btk inhibitor of Formula VII dosage will be 500 mg/m² and will be givenas a 10-minute infusion on Day 1 of each 14-day cycle. Folic acid andvitamin B12 supplementation, and dexamethasone (or equivalentcorticosteroid) prophylaxis will also be administered.

Variables

Efficacy: Tumor response rate will be defined as the number of patientswith documented partial response (PR) or complete response (CR) dividedby the number of patients qualified for tumor response analysis.Time-to-event analyses will be performed on the observed distributionsof time to objective progressive disease, progression-free survival(PFS), time to treatment failure (TtTF), and overall survival (OS) usingthe Kaplan-Meier (K-M) method. All patients with best overall responseof CR or PR will be analyzed for response duration by using the K-Mmethod.

Safety: Safety analyses will include adverse event (AE) rates, seriousAEs, vital signs, laboratory data, blood transfusions required, anddeaths. Toxicities using laboratory and nonlaboratory adverse eventswill be evaluated using the common terminology criteria for adverseevents (CTCAE, version 3.0).

Evaluation Methods

Statistical: The primary analysis will be to estimate the objective bestoverall response rate and its 95% confidence interval (CI). Medians foreach of the time-to-event endpoints, and time-to-event variables will beestimated using the K-M method. All estimates of treatment effects willbe conducted at a two-sided alpha level of 0.05, and CI for allparameters will be estimated were to be constructed using a 95% level.

Example 10: Breast Cancer Clinical Trial Length of Study

6 months

Objectives

The primary objective of this study will be to determine the objectiveresponse rate (ORR) for Btk inhibitors when administered every 2 weeksto patients with breast cancer. The secondary objectives of this studywill be to measure time-to-event variables including: time to objectivetumor response for responding patients (TtOR), duration of response forresponding patients, time to treatment failure (TtTF), time toprogressive disease (TtPD), progression-free survival (PFS), overallsurvival (OS); the toxicities of therapy.

Study Design

The study will be a multi-center, double-blind, randomized,placebo-controlled Phase 2 study. Tumor assessments will be repeatedevery 4 cycles (approximately 6 weeks). Patients will receive studytherapy for 12 treatments, or until tumor progression was documented,unacceptable toxicity was experienced, the patient withdrew consent, orthe patient is unable to fulfill the responsibilities of studyparticipation as determined by the treating physician or the qualifiedinvestigator. After study discontinuation, patients who have notprogressed will have tumor assessments performed approximately every 6weeks until disease progression. Once patients have disease progression,patients will enter a post-study follow-up period, and will be followedevery 12 weeks for 24 months for overall survival. Patients will also befollowed for ongoing or any new toxicities.

Diagnosis and Main Criteria for Inclusion:

Female patients aged 18 years or older

Histologically-confirmed ER-negative, progesterone receptor(PgR)-positive or PgR-negative, metastatic breast cancer

Cancer not Life-Threatening

No previous endocrine or cytotoxic treatment for metastatic breastcancer.

Study Drug, Dose, and Mode of Administration

Btk inhibitor of Formula VII dosage will be 1000 mg/m² and will be givenas a 60-minute infusion on Day 1 of each cycle. Folic acid and vitaminB12 supplementation, and dexamethasone (or equivalent corticosteroid)prophylaxis will also be administered.

Variables

Efficacy: Tumor response rate will be defined as the number of patientswith documented partial response (PR) or complete response (CR) dividedby the number of patients qualified for tumor response analysis.Time-to-event analyses will be performed on the observed distributionsof time to objective progressive disease, progression-free survival(PFS), time to treatment failure (TtTF), and overall survival (OS) usingthe Kaplan-Meier (K-M) method. All patients with best overall responseof CR or PR will be analyzed for response duration by using the K-Mmethod.

Safety: Safety analyses will include adverse event (AE) rates, seriousAEs, vital signs, laboratory data, blood transfusions required, anddeaths. Toxicities using laboratory and nonlaboratory adverse eventswill be evaluated using the common terminology criteria for adverseevents (CTCAE, version 3.0).

Evaluation Methods

Statistical: The primary analysis will be to estimate the objective bestoverall response rate and its 95% confidence interval (CI). Medians foreach of the time-to-event endpoints, and time-to-event variables will beestimated using the K-M method. All estimates of treatment effects willbe conducted at a two-sided alpha level of 0.05, and CI for allparameters will be estimated were to be constructed using a 95% level.

Example 11: Breast Cancer Clinical Trial

An MDA-MB-453 breast cancer xenograft was implanted under the skin of anude mouse. On a daily basis, administration of Compound 1 was effectedby intravenous administration at a level of (a) 50 mg/kg of mouseweight, or (b) 5 mg/kg of mouse weight. The volume of the tumorxenograft was monitored daily.

Example 12

Assays and Reagents

All the cells are purchased from ATCC and cultured as indicated from thesource; Antibodies are from Cell Signaling and Sata CluzBiotechnologies; Western blotting reagents from Life Technology(Invitrogen)

Cell proliferation assay with alamar blue: cells are plated at 10,000cells/well in 100 uL. After 3 day incubation at 370 C in 5% CO2incubator, alamar blue (Invitrogen 1:10) is added into each well andincubated for another 2.5-3 h. Read the plate at Ex/Em-545/590 nm.

Immunoblotting: Cells are washed once with cold PBS and lysed in 1×sample buffer (Invitrogen). The whole cell lysate is boiled, sonicated,and then loaded into 4-15% gradient SDS-PAGE gel. The proteins aretransferred to PVDF membrane. After probing with antibodies, thedetection is conducted using Odyssey spectrometer (Li-Cor).

Results

Breast cancer lines (BT-474, SK-BR3, MDA-MB-453 and UACC-893) which areHER2 amplified are sensitive to the growth inhibitory effect of ibrutini(see e.g., FIGS. 9a-9d ).

Ibrutinib induces apoptosis in BT-474 cells (see e.g., FIGS. 10a and 10b).

Ibrutinib inhibits tumor growth in a mouse MDA-MB-453 xenograft (seee.g. FIG. 11).

Breast cancer lines that are sensitive to ibrutinib are HER2/4-amplified(see e.g., FIGS. 12 and 13).

Ibrutinib is more potent than gefitinib in inhibiting MDA-MB-453 cellgrowth (see e.g., FIG. 14).

Ibrutinib shows similar activity with lapatinib, neratinib anddacomitinib (see e.g., FIGS. 16a, 16b, 17a, 17b, and 17c ).

1-14. (canceled)
 15. A method for treating sarcoma in a subjectcomprising administering to the subject a therapeutically effectiveamount of a compound having the structure:


16. The method of claim 15, wherein the sarcoma is selected from thegroup consisting of a sarcoma of the bone, a sarcoma of the cartilage, asarcoma of the fat, a sarcoma of the muscle, a sarcoma of the bloodvessels, a sarcoma of the connective tissue and a sarcoma of thesupportive tissue.
 17. The method of claim 15, wherein the sarcoma isselected from the group consisting of: chondrosarcoma, Ewing's sarcoma,malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, softtissue sarcomas, alveolar soft part sarcoma, angiosarcoma, cystosarcomaphylloides, dermatofibrosarcoma, desmoid tumor, epithelioid sarcoma,extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma,hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma,neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma.
 18. Themethod of claim 15, wherein survival of the subject is prolonged. 19.The method of claim 15, wherein the method further comprisesadministering an additional cancer agent.
 20. The method of claim 19,wherein the additional cancer agent is ifosfamide or doxorubicin.